(Harold H Trimm) Inorganic Chemistry Reactions

Trimm
Inorganic Chemistry
Reactions, Structure and Mechanisms Research Progress in Chemistry
Inorganic chemistry is the study of all chemical compounds except those containing carbon, which
is the field of organic chemistry. There is some overlap since both inorganic and organic chemists
traditionally study organometallic compounds. Inorganic chemistry has very important
ramifications for industry. Current research interests in inorganic chemistry include the discovery
Inorganic Chemistry
of new catalysts, superconductors, and drugs to combat disease. This new volume covers a
diverse collection of topics in the field, including new methods to detect unlabeled particles, Reactions, Structure and Mechanisms
measurement studies, and more.
Inorganic Chemistry
Reactions, Structure and Mechanisms
About the Editor
Dr. Harold H. Trimm was born in 1955 in Brooklyn, New York. Dr. Trimm is the chairman of the
Chemistry Department at Broome Community College in Binghamton, New York. In addition, he is
an Adjunct Analytical Professor, Binghamton University, State University of New York,
Binghamton, New York.
He received his PhD in chemistry, with a minor in biology, from Clarkson University in 1981 for his
Harold H. Trimm, PhD
work on fast reaction kinetics of biologically important molecules. He then went on to Brunel Editor
University in England for a postdoctoral research fellowship in biophysics, where he studied the
molecules involved with arthritis by electroptics. He recently authored a textbook on forensic
science titled Forensics the Easy Way (2005).
Other Titles in the Series

• Analytical Chemistry: Methods and Applications
• Organic Chemistry: Structure and Mechanisms
• Physical Chemistry: Chemical Kinetics and Reaction Mechanisms
Related Titles of Interest

• Environmental Chemistry: New Techniques and Data
• Industrial Chemistry: New Applications, Processes and Systems
• Recent Advances in Biochemistry
ISBN 978-1-926692-59-3
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Apple Academic Press

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Inorganic Chemistry
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Research Progress in Chemistry
Inorganic Chemistry
Harold H. Trimm, PhD, RSO

Chairman, Chemistry Department, Broome Community College;
Adjunct Analytical Professor, Binghamton University,
Binghamton, New York, U.S.A.

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Contents
Introduction 9
1. Inorganic Polyphosphate Modulates TRPM8 Channels 11
Eleonora Zakharian, Baskaran Thyagarajan, Robert J. French,
Evgeny Pavlov and Tibor Rohacs
2. On the Origin of Life in the Zinc World: 1. Photosynthesizing, 36
Porous Edifices Built of Hydrothermally Precipitated Zinc Sulfide
as Cradles of Life on Earth
A. Y. Mulkidjanian
3. On the Origin of Life in the Zinc World: 2. Validation of the 103
Hypothesis on the Photosynthesizing Zinc Sulfide Edifices as
Cradles of Life on Earth
A. Y. Mulkidjanian and M. Y. Galperin
4. Evaluation of New Inorganic Sorbents for Strontium and 165
Actinide Removal from High-Level Nuclear Waste Solutions
D. T. Hobbs, M. Nyman, D. G. Medvedev, A. Tripathi and
A. Clearfield
5. Origin of Selectivity in Tunnel Type Inorganic Ion Exchangers 170
Abraham Clearfield, Akhilesh Tripathi, Dmitri Medvedev,
Jose Delgado and May Nyman
6 Inorganic Chemistry: Reactions, Structure and Mechanisms
6. Development of Inorganic Membranes for Hydrogen Separation 173

Brian L. Bischoff and Roddie R. Judkins
7. Nickel (II), Copper (II) and Zinc (II) Complexes of 183
9-[2- (Phosphonomethoxy)ethyl]-8-azaadenine (9,8aPMEA),
the 8-Aza Derivative of the Antiviral Nucleotide Analogue
9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA). Quantification
of Four Isomeric Species in Aqueous Solution
Raquel B. Gómez-Coca, Antonín Holy, Rosario A. Vilaplana,
Francisco González-Vilchez and Helmut Sigel
8. Inorganic Speciation of Dissolved Elements in Seawater: 205
The Influence of Ph on Concentration Ratios
Robert H. Byrne
9. A Novel Method to Detect Unlabeled Inorganic Nanoparticles 217
and Submicron Particles in Tissue by Sedimentation Field-Flow
Fractionation
Cassandra E. Deering, Soheyl Tadjiki, Shoeleh Assemi, Jan D. Miller,
Garold S. Yost and John M. Veranth
10. Chemical Speciation of Inorganic Compounds Under 234
Hydrothermal Conditions
M. M. Hoffmann, J. L. Fulton, J. G. Darab, E. A. Stern, N. Sicron,
B. D. Chapman and G. Seidler
11. Measurements of Particle Masses of Inorganic Salt Particles 239
for Calibration of Cloud Condensation Nuclei Counters
M. Kuwata and Y. Kondo
12. Crystal Structure of [Bis(L-Alaninato)Diaqua]Nickel(II) Dihydrate 263
Awni Khatib, Fathi Aqra, David Deamer and Allen Oliver
13. Mean Amplitudes of Vibration of the IF8 − Anion 273
Enrique J. Baran
14. Mechanistic Aspects of Osmium(VIII) Catalyzed Oxidation 278
of L-Tryptophan by Diperiodatocuprate(III) in Aqueous Alkaline
Medium: A Kinetic Model
Nagaraj P. Shetti, Ragunatharaddi R. Hosamani and
Sharanappa T. Nandibewoor
Contents 7
15. Kinetic and Mechanistic Studies on the Reaction of 286

DL-Methionine with [(H2O)(tap)2RuORu(tap)2(H2O)]2+ in
Aqueous Medium at Physiological pH
Tandra Das A. K. Datta and A. K. Ghosh
16. Molybdenum and Tungsten Tricarbonyl Complexes of Isatin 296
with Triphenylphosphine
M. M. H. Khalil and F. A. Al-Seif
17. Synthesis and Characterization of Biologically Active 303
10-Membered Tetraazamacrocyclic Complexes of Cr(III),
Mn(III), and Fe(III)
Dharam Pal Singh, Vandna Malik and Ramesh Kumar
18. Antifungal and Spectral Studies of Cr(III) and Mn(II) Complexes 312
Derived from 3,3'-Thiodipropionic Acid Derivative
Sulekh Chandra and Amit Kumar Sharma
Index 321
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Introduction
Chemistry is the science that studies atoms and molecules along with their prop-
erties. All matter is composed of atoms and molecules, so chemistry is all encom-
passing and is referred to as the central science because all other scientific fields
use its discoveries. Since the science of chemistry is so broad, it is normally broken
into fields or branches of specialization. The five main branches of chemistry are
analytical, inorganic, organic, physical, and biochemistry. Chemistry is an ex-
perimental science that is constantly being advanced by new discoveries. It is the
intent of this collection to present the reader with a broad spectrum of articles in
the various branches of chemistry that demonstrates key developments in these
rapidly changing fields.
Inorganic chemistry is the study of all chemical compounds except those con-
taining carbon, which is the field of organic chemistry. There is some overlap,
since both inorganic and organic chemists traditionally study organometallic
compounds, such as the cancer fighting drug cisplatin. Inorganic chemistry is
very important in industry. The size of a country’s manufacturing output is tra-
ditionally measured by its production of the inorganic chemical sulfuric acid,
which is the basis for many industrial processes. Current advances in inorganic
chemistry include the discovery of new catalysts, superconductors, and drugs to
combat disease. Much of the green revolution in farming, which allows us to feed
the earth’s population, is based on the inorganic chemist’s ability to produce fertil-
izer from cheap raw materials.
The chapters included within this book will ensure that the reader stays cur-
rent with the latest methods and applications in this important field.
— Harold H. Trimm, PhD, RSO

Inorganic Polyphosphate
Modulates TRPM8 Channels
Eleonora Zakharian, Baskaran Thyagarajan, Robert J. French,

Evgeny Pavlov and Tibor Rohacs
Abstract
Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of
phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms
complexes and modulates activities of many proteins including ion channels.
Here we investigated the role of polyP in the function of the transient recep-
tor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp
and fluorescent calcium measurements we demonstrate that enzymatic break-
down of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in
human embryonic kidney and F-11 neuronal cells expressing TRPM8. We
demonstrate that the TRPM8 channel protein is associated with polyP. Fur-
thermore, addition of scPPX1 altered the voltage-dependence and blocked the
activity of the purified TRPM8 channels reconstituted into planar lipid bi-
layers, where the activity of the channel was initiated by cold and menthol
in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2). The
biochemical analysis of the TRPM8 protein also uncovered the presence of

poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with pol-
yP. We conclude that the TRPM8 protein forms a stable complex with polyP
and its presence is essential for normal channel activity.
Introduction
TRPM8 is a member of the transient receptor potential (TRP) channel family of
the melastatin subgroup, which is thought to be a major sensor for a wide range
of cold temperatures in the peripheral nervous system [1], [2], [3]. TRPM8 is
activated by low temperatures in the range of 8–26°C and a number of chemical
compounds such as menthol, icilin, eucalyptol, geraniol and linalool [4], [5], [6].
Several other factors, such as voltage [7], [8], pH [8], lysophospholipids and fatty
acids [9], [10] also modulate TRPM8 activity.
A major intracellular factor that is required for the channels activity of TRPM8
is phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) [11], [12]. PtdIns(4,5)
P2 regulation is a common property of many TRP channels [13], [14], [15] and
several other ion channels from different families [16], [17], [18], [19]. In general
the dynamic changes in the levels of plasma membrane phosphoinositides have
been shown to play regulatory roles in many ion transporting systems [20], [21],
[22]. TRP channel functions could also be modified by inorganic polyphosphates
apart from phosphoinositides. Recently it has been shown that TRPA1 channels
can be activated by pungent chemicals only in the presence of inorganic poly-
phosphates [23].
Inorganic polyphosphate (poly P) is a polymer of tens or hundreds of phos-
phate residues linked by high-energy anhydride bonds as in ATP. PolyP plays cen-
tral roles in many general physiological processes, acting as a reservoir of energy
and phosphate, as a chelator of metals, as a buffer against alkali. In microorgan-
isms it is essential, for example, for physiological adjustments to growth condi-
tions as well as to stress response [24]. Polyphosphates are present in all higher
eukaryotic organisms, where they likely play multiple important roles [25], [26],
[27]. In higher eukaryotes, polyP contributes to the stimulation of mammalian
target of rapamycin, involved in the proliferation of mammary cancer cells [28]
and regulates mitochondrial function [29]. However, many aspects of polyP func-
tion in these organisms remain to be uncovered.
PolyP is also believed to be an important participant in ion transport. PolyP, in
association with a solvating amphiphilic polymer of R-3-hydroxybutyrate (PHB),
can form ion channels with high selectivity for cations [30]. Channel forming
polyP/PHB Ca2+ complexes have been found in bacterial and mitochondrial
Inorganic Polyphosphate Modulates TRPM8 Channels 13
membranes [30], [31], [32]. Furthermore, polyP and PHB are associated with a
variety of membrane proteins, including several bacterial ion channels and might
be required for their normal functioning [33], [34].
In the present study, we demonstrate that TRPM8 expressed in HEK-293 and
F-11 neuronal cells is associated with polyP and PHB, and that polyP serves as
crucial regulator of TRPM8 channel function.
Methods
Cell Culture
HEK-293 cells were maintained in minimal essential medium (MEM) solution
(Invitrogen, San Diego, CA) supplemented with 10% fetal bovine serum (Invit-
rogen) and 1% penicillin/streptomycin. The rat TRPM8 tagged with the myc
epitope on the N-terminus, scPPX1, GFP in pCDNA3 vectors were transfected
using the Effectene reagent (Qiagen, Chatsworth, CA). Two different TRPM8
stable cell lines were developed: one with TRPM8 myc-tagged on the N-terminus
(TRPM8-myc), and one with TRPM8 tagged with myc on the N-terminus and
with 6His residues on the C-terminus (TRPM8-his). These stable cell lines were
obtained using the following procedure: HEK-293 cells were treated with dif-
ferent concentration of G418 to determine killing concentration of G418 (Sig-
ma, St. Louis, MO). Then cells were transfeced with lineralized TRPM8-myc or
TRPM8-his cDNA using effectene transfection reagent. 24 hours after transfec-
tion, cells were treated with 1 mg/ml G418 containing MEM supplemented with
10% FBS and antibiotics. After 7 days, single cells were selected from clonal rings
and these were seeded on 24 well plates for further propagation of each single
clone. The individual clones were pooled into a single culture and propagated in
the presence of 400 µg/ml G418. Forty eight hrs before the experiment, cells were
split into MEM supplemented with FBS and antibiotics but without G418.
F-11 cells were cultured in DMEM/F12 medium +20% FBS, 0.2 mM L-glu-
tamine, 100 µM sodium hypoxanthine, 400 nM aminopterin, 16 µM thymidine
(HAT supplement), and penicillin/streptomycin at 37°C (the cells were kindly
provided by Dr. S.E. Gordon, University of Washington).
Mammalian Electrophysiology
Whole-cell patch clamp measurements were conducted 36–72 h after propaga-
tion of the TRPM8 stable cell lines or transient transfection of target clones. The
extracellular solution contained (in mM) 137 NaCl, 5 KCl, 1 MgCl2, 10 glucose,
and 10 HEPES, pH 7.4. Borosilicate glass pipettes (World Precision Instruments,
Sarasota, FL) of 2–4 MΩ resistance were filled with a solution containing (in
mM) 135 K-gluconate, 5 KCl, 5 EGTA, 1 MgCl2, and 10 HEPES, pH 7.2. For
the experiments the pipette solution was supplemented with 2 mM ATP. After
formation of GΩ-resistance seals, whole-cell configuration was established, and
currents were measured at a holding potential of −60 mV, using an Axopatch
200B amplifier (Molecular Devices, Union City, CA). Current-voltage ramp rela-
tions were recorded using voltage ramps from −100 to +100 mV with a duratron
of 0.8 s. Data were collected and analyzed with the pClamp 9.0 software. Mea-
surements were performed at room temperature (~22°C).
Intracellular Ca2+ Measurements

The extracellular solution used in ratio-metric [Ca2+]i measurements contained
(in mM) 137 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, 10 Glucose and 10 Hepes,
pH 7.4. Cells were incubated with 2 µM Fura-2 acetoxymethyl ester (Tef Labs
Austin, TX) for 30 min. at room temperature. The fluorescence signals of single
cells were measured using alternating excitation at 340 and 380 nm and emission
was detected at 510 nm. The ratio of fluorescence (340/380) was plotted against
time. The measurements were performed using a Photon Technology Interna-
tional (PTI) (Birmingham, NJ) photomultiplier based system mounted on an
Olympus IX71 microscope, equipped with a DeltaRAM excitation light source,
or with a Ratiomaster 5 Imaging System (PTI) equipped with a Cool-snap HQ2
(Roper) Camera.
Preparation of the TRPM8 Protein

HEK-293 cells stably expressing TRPM8 were grown to 70–80% confluence,
washed and collected with cold PBS. Cells were harvested and resuspended in
0.25 M sucrose-1 mM triethanolamine (TEA) HCl, with addition of a protease
inhibitor cocktail (Roche, Indianapolis, IN), pH 7.4. Plasma membranes were
isolated by differential centrifugation. The TRPM8 protein was extracted from
plasma membranes with (in mM) 137 NaCl, 5 KCl, 1 MgCl2, 10 Glucose and
10 Hepes, pH 7.4, in presence of 1% Nonidet P40 (Roche) and 0.5% dodecyl-
maltoside (DDM) (Roche), and the protease inhibitors, upon incubation at 4°C
on a shaker with gentle agitation for 2 h. This suspension was further centrifuged
for 1 h. at 100,000 g. The supernatant was concentrated with 100 K Amicon
centrifuge filters (Millipore-Fisher) and purified by gel-filtration chromatography
on Sephacryl S-300 column (1.6×60 cm GE Healthcare, Piscataway, NJ) equili-
brated with the same buffer containing 2 mM DDM. All steps of purification
were performed at 4°C. After elution from the column, protein fractions were
concentrated to a final concentration of 12 µg/ml and analyzed by Western blot

analysis with anti-c-Myc IgG antibodies (Sigma). For some of the planar lipid
bilayer experiments, in order to improve the stability of the artificial membranes
with the incorporated protein, we also purified TRPM8 from the TRPM8-his
stable cell line. This modification allowed us to include into the procedure de-
scribed above an additional step of purification with ion-affinity chromatography
using Ni-NTA beads (Qiagen).
SDS-PAGE
Proteins were electrophoretically separated on 7.5 or 10% SDS-PAGE (Bio-Rad,
Hercules, CA) using Tris-glycine sodium dodecyl sulfate (SDS) buffer (Bio-Rad)
at a constant voltage of 180 V. The electrophoresis buffer for the native gels did
not contain SDS. Protein bands were visualized by staining with Coomassie bril-
liant blue R-250. For Western blot analysis, protein was transferred onto nitro-
cellulose membranes (Bio-Rad) in 10 mM CAPS, 0.07% SDS buffer at 30 V
overnight. The TRPM8 protein was detected with anti-Myc-IgG antibodies.
Determination of PolyP
PolyP was visualized on the native 7.5% or 10% polyacrylamide Ready Gels from
Bio-Rad (Helcules, CA, USA). Electrophoresis was performed at 100 V for 1–1.5
h. Gels were incubated for 1 h. in fixative solution consisting of 25% methanol
/ 5% glycerol, stained for 30 min with 0.05% -o-toluidine blue and destained in
a fixative for 2 hours. To eliminate polyP, the samples were treated with 2 µg/ml
exopolyphosphatase of Saccharomyces cerevisiae scPPX1.
Determination of PHB
PHB was detected by Western blot analysis with anti-PHB IgG raised in rabbits
to a synthetic 8-mer of R-3-hydroxybutyrate (kindly provided by Dr. Rosetta N.
Reusch).
Planar Lipid Bilayer Measurements

Planar lipid bilayers were formed from a solution of synthetic 1-palmitoyl-2-
oleoyl-glycero-3-phosphoco line (POPC) and 1-palmitoyl-2-oleoyl-glycero-3-
phosphoethanolaminein (POPE, Avanti Polar Lipids, Birmingham, AL) in ratio
3:1 in n-decane (Aldrich). The solution was used to paint a bilayer in an aper-
ture of ~150 µm diameter in a Delrin cup (Warner Instruments, Hamden, CT)
between symmetric aqueous bathing solutions of 150 mM KCl, 20 mM Hepes,

pH 7.2, at 22°C. All salts were ultrapure (>99%) (Aldrich). Bilayer capacitances
were in the range of 50–75 pF. After the bilayers were formed, 0.2–0.5 µl of
the TRPM8 micellar solution (2 µg/ml) was added to the cis compartment with
gentle stirring. Unitary currents were recorded with an integrating patch clamp
amplifier (Axopatch 200A, Axon Instruments). The trans solution (voltage com-
mand side) was connected to the CV 201A head stage input, and the cis solution
was held at virtual ground via a pair of matched Ag-AgCl electrodes. Currents
through the voltage-clamped bilayers (background conductance <3 pS) were fil-
tered at the amplifier output (low pass, −3 dB at 10 kHz, 8-pole Bessel response).
Data were secondarily filtered at 50 Hz through an 8-pole Bessel filter (950 TAF,
Frequency Devices) and digitized at 1 kHz using an analog-to-digital converter
(Digidata 1322A, Axon Instruments), controlled by pClamp9 software (Axon
Instruments). Single-channel conductance events, all points’ histograms, open
probabilities and other parameters were identified and analyzed using the Clamp-
fit9 software (Axon Instruments).
Temperature Studies
For temperature studies, a Delrin cuvette was seated in a bilayer recording cham-
ber made of a thermally conductive plastic (Warner Instruments). The chamber
was fitted on a conductive stage containing a pyroelectric heater/cooler. Deion-
ized water was circulated through this stage, pumped into the system to remove
the heat generated. The pyroelectric heating/cooling stage was driven by a tem-
perature controller (CL-100, Warner Instruments). The temperature of the bath
was monitored constantly with a thermoelectric device in the cis side, i.e. the
ground side of the cuvette. Although there was a temperature gradient between
the bath solution and conductive stage, the temperature within the bath could be
reliably controlled within ±0.5°C.
Results
Inhibition of TRPM8 Currents and Ca2+ Signals by
Exopolyphophatase
In developing the methods for our studies we have used an enzymatic approach
with application of Saccharomyces cerevisiae exopolyphosphatase X (scPPX1).
ScPPX1 is an effective polyphosphatase that possesses high substrate specific-
ity and hydrolyses orthophosphates from polyP chains of varied lengths, but
not from ATP, pyrophosphate and trimetaphosphate [35]. In order to detect a
possible effect of polyP on TRPM8 we conducted a number of experiments with

application, or expression, of scPPX1 in HEK-293 cells. In whole-cell patch
clamp experiments, dialysis of purified scPPX1 through the patch pipette into
cells transiently transfected with TRPM8 significantly inhibited menthol-induced
currents in a time period of 3–5 min of treatment with scPPX1 (Fig. 1A–C). The
concentration of scPPX1 in the pipette solution (2.3 µg/ml) was sufficient to
observe inhibition within the tested time. We found that higher concentrations
of scPPX1 were toxic for the cells. In control experiments the menthol-activated
TRPM8 currents were found to be 1.8±0.21 and 1.77±0.25 nA (n = 8) for the
first and the second pulses of menthol application, while the values were found
to be 0.83±0.18 and 0.68±0.16 nA (n = 5) in scPPX1 dialyzed cells. The record-
ings were obtained at holding potential of −60 mV. The results are summarized in
figure 1C. All the errors are expressed as SEM.
Figure 1. Inhibition of TRPM8 currents by scPPX1 in whole-cell patch clamp. Upper panels: Whole-cell patch
clamp measurements of menthol-induced currents were performed at −60 mV in the whole-cell configuration
on HEK cells expressing TRPM8, in nominally Ca2+-free solution (NCF), to avoid desensitization. Menthol
pulses (500 µM) were applied in the first 3–5 min after establishment of whole-cell configuration: HEK-293
cells were transiently transfected with TRPM8 (0.4 µg) and co-transfected with GFP clone (0.2 µg) to allow
detection of transfected cells. Panel A: the control. Panel B: the pipette solution was supplemented with 2.3 µg/
ml scPPX1. Midle panels: Whole-cell patch clamp was performed on HEK-293 TRPM8 stable cell line, which
was transiently transfected with GFP (0.2 µg) alone (panel D) or with scPPX1 clone (0.4 µg) and GFP (0.2 µg)
(panel E). The summaries are shown in panel F. The protocol of experiment is the same as for the measurements
in the upper panel. Lower panels: Current/Voltage relationships of TRPM8 channels obtained in whole-cell
patch clamp performed at −100 +100 mV voltage ramps for HEK-293 TRPM8 stable cell line, which was
transiently transfected with GFP (0.2 µg) alone (panel G) or with scPPX1 clone (0.4 µg) and GFP (0.2 µg)
(panel H). The summaries are shown in panel I at −100 and +100 mV.
We next tested the effect of scPPX1 by transiently transfecting scPPX1-pcD-

NA (0.4 µg) into HEK-293 cells stably expressing TRPM8 (TRPM8-HEK293).
Cells were co-transfected with GFP (0.2 µg) to allow detection of transfected cells
(Fig. 1D–F). Control experiments were performed in TRPM8-HEK293 cells
expressing GFP alone. In controls, the values of menthol-induced currents ob-

tained at −60 mV were 0.94±0.12 and 0.915±0.122 nA (n = 7) and in scPPX1
expressing cells the values were found to be 0.054±0.001 and 0.051±0.008 nA (n
= 8), for the first and the second pulses, respectively.
The current-voltage relationships of TRPM8 channels in the control cells and
scPPX1-expressing cells are demonstrated in Figure 1G–I. We found that inward
currents of TRPM8 exhibit more profound inhibition by scPPX1 (~83%) than
outward currents (~65%).
Next we monitored intracellular Ca2+ signals induced by menthol in single
TRPM8-HEK293 cells co-expressing scPPX1. Figure 2 (A–C) shows representa-
tive experiments, where 50 and 500 µM menthol were added to single cells in the
presence of 1.8 mM Ca2+. Menthol-evoked Ca2+ signals were observed as an in-
crease in the fluorescence intensity ratio of fura-2 (340/380). We found that men-
thol-induced intracellular Ca2+ signals were significantly inhibited (p≤0.005) in
cells with co-expressed scPPX1 (0.156±0.085, n = 9) in comparison to the control
cells (0.9±0.2, n = 6) (Fig. 2A–C). Further we performed analogous measurements
in F-11 neuronal cells that were derived from rat dorsal root ganglion neurons.
F-11 cells are used as a model for DRG neurons to study sensory TRP channels
in a system resembling their native environment [36]. In our experiments, F-11
cells were transiently transfected with TRPM8 (0.4 µg) alone or together with
scPPX1 (0.4 µg), and menthol-induced Ca2+ signals were subsequently analyzed
(Fig. 2D–F). Similarly to the HEK cells expression system, we found that men-
thol-evoked Ca2+-entry was significantly inhibited (p≤0.005) in F-11 cells with
co-expressed scPPX1 (0.105±0.029, n = 12) in comparison to the control cells
expressing TRPM8 channels alone (0.85±0.119, n = 11) (Fig. 2D–F).
Figure 2. Inhibition of TRPM8 activity by scPPX1 in intracellular Ca2+ measurements. Upper panels:
Fluorescence measurements of intracellular Ca2+ concentration were performed on HEK-293 TRPM8 stable
cell lines with transiently transfected GFP (0.2 µg) alone (panel A) or together with the scPPX1 clone (0.4 µg)
(panel B). The summaries of averaged menthol responses are represented in panel C. Lower panels: Fluorescence
measurements of intracellular Ca2+ signals were performed on F-11 neuronal cells with transiently transfected
TRPM8 (0.4 µg) and GFP (0.2 µg) (panel D) or together with the scPPX1 clone (0.4 µg) (panel E). The
summaries of averaged menthol responses are represented in panel F.
In order to test whether the significant inhibition of TRPM8 channel activity

by scPPX1 detected in the patch clamp and Ca2+ measurements is due to the
alteration of the levels and/or localization of the TRPM8 protein, we performed
Western blot and immunocytochemical analyses on HEK-293 cells expressing
TRPM8 alone, or with the enzyme (see Methods S1). In the immunocytochem-
istry experiments, TRPM8 showed both intracellular and plasma membrane lo-
calization, consistent with earlier studies [37]. Co-expression of scPPX1 did not
alter the localization of TRPM8 (Fig. S1A) and did not decrease the amount of
the protein as detected with Western blot (Fig. S1B).
TRPM8 channels require PtdIns(4,5)P2 for activity. In order to ensure that
expression of scPPX1 did not affect PtdIns(4,5)P2 levels in the cells, we have
monitored the distribution of the GFP-tagged PH-domain of phospholipase C
δ1 in control HEK cells, and in cells transfected with scPPX1. Co-expression of
scPPX1 did not change the plasma membrane localization of the GFP tagged PH
domain, indicating that plasma membrane PtdIns(4,5)P2 levels were not signifi-
cantly altered (data not shown).
Inorganic Polyphosphate and Polyhydroxybutyrate Associate

with TRPM8
The dramatic inhibition of TRPM8 channel activity by scPPX1 led us to inves-
tigate whether polyP is associated with the protein. For our studies of the bio-
chemical and biophysical properties of TRPM8, we purified the protein from the
HEK-293 cell line stably expressing TRPM8. Plasma membranes were isolated by
differential centrifugation with subsequent extraction of the TRPM8 protein with
1% Nonidet and 0.5% dodecylmaltoside (DDM) (see materials and methods).
These conditions were favorable for harvesting a large amount of TRPM8 from
the plasma membranes of cells stably expressing the protein. For control, the same
extraction conditions were applied to the plasma membranes of HEK-293 cells
not expressing TRPM8, where no TRPM8 protein was detected with anti-Myc
IgG (Fig. 3, lane 1). In order to receive homogeneous fraction of the protein,
TRPM8 was further purified by gel-filtration chromatography on Sehpacryl-300
column in 134 NaCl mM, 5 KCl mM, 1 MgCl2 mM and 10 Hepes mM, pH 7.4,
containing 2 mM DDM. After elution from the column, fractions of the protein
were concentrated in amicon-100 centrifuge tubes and analyzed by Western blot
with anti-Myc IgG (Fig. 3). Analogously, we have tested a number of other de-
tergents, including decylmaltoside, LDAO, octylglucoside, triton-X100, etc. for
TRPM8 extraction and purification purposes, however only DDM resulted in a
relatively high yield of purified protein and supported the stability of a tetrameric
form of TRPM8, which was identified by gel-filtration chromatography and elec-
trophoresis on the native gels.
Figure 3. Western blots of TRPM8 protein derived from expression in HEK-293 cell lines. TRPM8 protein
samples were separated on a 10% SDS-PAGE and blotted on nitrocellulose membranes overnight in the presence
of CAPS buffer (pH 11.1). Immunodetection was revealed by chemiluminescence. Lanes 1–3 probed with anti-
Myc-IgG: Lane 1 – plasma membrane fractions of HEK-293 cells not expressing TRPM8; Lane 2 – plasma
membrane extracts of cells stably expressing TRPM8; Lane 3 – TRPM8 protein purified on Sephacryl-300
gel-filtration chromatography. Lane 4 – Coomassie blue staining of purified TRPM8. Samples were heated for
5 min. at 70°C before loading.
The presence of polyP in tetramers of TRPM8 was detected by its metachro-

matic reaction to the cationic dye, o-toluidine blue. PolyP of >5 residues causes
a shift in the absorption on maximum of o-toluidine blue toward shorter wave-
lengths, i.e., from 630 nm (blue) to 530 nm (violet-red) [38]. PolyP stains a
distinctive reddish-purple color on PAGE gels (Fig. 4A, lane 2). The identity of
polyP was confirmed by its complete degradation when TRPM8 was incubated
with 2 µg/ml scPPX1 (Wurst et al., 1995) for 3 h at 37°C before loading on the
gel (Fig. 4A, lane 3). The presence of TRPM8 in lanes 2 and 3 of the gel was
confirmed by re-staining the gel with Coomassie blue (lanes 4 and 5). The protein
and polyP detected on the native gels migrate at an apparent molecular weight of
490–500 kDa, which corresponds to the molecular weight of TRPM8 in the te-
trameric form. The association of polyP with the TRPM8 protein was confirmed
after each protein purification procedure. A total of 12 native PAGE experiments
were performed for detection of polyP.
Figure 4. A. Detection of polyP associated with the TRPM8 protein. TRPM8 was separated on native PAGE
to preserve its migration in the tetrameric form. Lane 1 – standards ladder (The High-Mark Pre-stained High
Molecular Weight Protein Standards, Invitrogen); Lane 2 – purified TRPM8 sample with o-toluidine blue stain
of native PAGE gel; Lane 3 – o-toluidine blue stain of native PAGE gel of the same TRPM8 sample treated with
1 µl scPPX1 (2 µg/ml) for 3 h. before loading: Lane 4 and 5 are lanes 2 and 3 re-stained with Coomassie blue. B.
Detection of PHB in TRPM8 in Western blot. Lane 1 – purified TRPM8 protein detected with antiMyc_IgG;
Lane 2 – Western blot of purified TRPM8 probed with anti-PHB-IgG. Samples were heated for 5 min. at 70°C
before loading.
Association of polyP with proteins has frequently been found to be mediated

by PHB, which is known to “solvate” metal cation salts of polyP [30], [39], [40].
We observed that PHB was also associated with TRPM8, which was detected by
Western Blot analysis using anti-PHB IgG [41] raised in rabbits to a synthetic
8-mer of R-3-hydroxybutyrate (Fig. 4B, lane 2).
Inhibition of TRPM8 Channel Activity by scPPX1 in Planar

Lipid Bilayers
The whole cell patch clamp experiments and intracellular Ca2+ measurements
demonstrated that depletion of polyP by the exopolyphosphatase scPPX1 inhib-
ited TRPM8 currents and Ca2+-entry. To understand whether the effect of polyP
is direct or indirect on the TRPM8 channel protein, we examined the single chan-
nel properties of TRPM8 incorporated in planar lipid bilayers and the effect of
subsequent treatment of the protein with scPPX1. The purified TRPM8 protein
derived in dodecylmaltoside (DDM) micelles was incorporated into lipid mi-
celles consisting of a mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocoline
and 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolaminein POPC/POPE (3:1,
v/v), and then into planar lipid bilayers of the same lipid composition between
aqueous solutions of 150 mM KCl, 0.2 mM MgCl2 in 20 mM Hepes, pH 7.2.

The presence of Mg2+ in the experimental solution was required to sustain normal
channel activity of TRPM8 with optimal concentration of 0.2 mM. Higher con-
centrations of Mg2+ (≥2 mM) evoked an inhibition of TRPM8 currents. We also
found that the presence of Mg2+ was necessary during the protein purification.
In the absence of this cation the tetramers of TRPM8 would disintegrate into
the monomers, and that in its turn would cause polyP dissociation. To confirm
the stability of TRPM8 in tetrameric form and the presence of polyP the native
PAGE were performed after each protein purification.
In order to stimulate channel activity we supplemented the experimental con-
ditions with menthol and/or PtdIns(4,5)P2. All experiments were conducted at
room temperature (~22°C). The representative current traces of TRPM8 channels
in planar lipid bilayers are given in Figure 5. No channels were observed when
TRPM8 alone was incorporated in the lipid bilayers (Fig. 5, n = 13). However,
addition of 2 µM of the short acyl-chain dioctanoyl (diC8) PtdIns(4,5)P2 re-
sulted in rare burst openings of TRPM8 (Po<0.001, n = 12), which was followed
by full opening of the channels upon addition of 500 µM menthol (Po = 0.9±0.1,
n = 11) (Fig. 5, upper trace). No TRPM8 openings were detected when men-
thol was added first, and fully open channels were observed when 2 µM diC8
PtdIns(4,5)P2 was supplemented into the bilayer (Po = 0.9±0.1, n = 17) (Fig. 5,
lower trace).
Figure 5. Activation of TRPM8 channels in Planar Lipid Bilayers by menthol and PtdIns(4,5)P2. Representative
single-channel current recordings of TRPM8 channels incorporated in planar lipid bilayers formed from POPC/
POPE (3:1) in n-decane, between symmetric bathing solutions of 150 mM KCl, 0.2 mM MgCl2 in 20 mM
Hepes buffer, pH 7.4 at 22°C. 0.2–0.5 µl of 0.2 µg/ml TRPM8 protein (isolated from the plasma membrane of
HEK-293 cells stably expressing TRPM8) was incorporated in POPC/POPE micelles, which were added to the
cis compartment (ground). Clamping potential was +60 mV. Data were filtered at 50 Hz. Upper and lower traces
consist of three segments with additions of components as indicated in the figure: 2 µM of diC8 PtdIns(4,5)P2
and 500 µM of menthol were added to both compartments. The current recordings are representative of a total
of 22 independent experiments for the upper traces and 12 independent experiments for the lower traces.
All the following bilayer experiments were conducted in presence of 1%

1,2-dipalmitoyl (diC16) PtdIns(4,5)P2, which resulted in higher stability of the
planar lipid bilayers in comparison to the short chain diC8 PtdIns(4,5)P2. No
menthol-activated channels were observed in presence of PtdIns(4,5)P2 on plas-
ma membrane fractions from HEK-293 cells not expressing TRPM8, total 11
experiments were conducted from three different plasma membrane preparations
(data not shown).
After the conditions for obtaining the channels in lipid bilayers were estab-
lished, we found that TRPM8 demonstrated different open probability and gat-
ing modes for current flowing in outward and inward directions. A single-channel
current-voltage relationship, and open probabilities are presented in Fig. 6 (A–C).
Channels were obtained in the presence of 1% diC16 PtdIns(4,5)P2 and 500 µM
menthol. Outward currents exhibited mean slope conductance values of 72±12
pS, and Po of ~0.89 at 100 mV (n = 11, number of events analyzed = 2,811), and
inward currents were observed in two conductance states with main conductance
level of 42±6 pS and Po of ~0.4 (at −100 mV) and rarely detected burst open-
ings of a subconductance state with mean conductance of 30±3 pS (Po≤0.001),
which would step to the fully open magnitude (72 pS) of the channels (n = 10,
number of events analyzed = 1,908). The observed value of the mean conductance
(72 pS at 22°C) is similar to that previously reported [5], [42]. The orientation
of the channels incorporated in the lipid bilayer was determined by outward rec-
tification inherent to TRPM8, and poly-lysine block [12]. As previously shown,
poly-lysine blocks TRPM8 currents from the cytoplasmic side of the channel.
Consistent with this, 30 µg/ml of poly-lysine added to the bath solution did not
significantly inhibit Ca2+ signals evoked by 500 µM menthol in cells expressing
TRPM8 (data not shown).
Next we investigated TRPM8 channel activity under various menthol con-
centrations and determined the menthol dose response on the single channel level
(Fig. 6D). We found that menthol at different concentrations affects TRPM8
activity by mainly altering the open probability of the channel (Fig. 6D). In the
figure 6D values of Po observed at 100 mV were plotted against menthol concen-
trations, total 36 experiments were conducted and numbers of events analyzed
for each menthol concentration were in a range of 400–1500. We also studied the
cold sensitivity of TRPM8 reconstituted into planar lipid bilayers. Figure 7 dem-
onstrates representative current traces of TRPM8 in planar lipid bilayers activated
by lowering temperature from 23°C to 16°C. Total 12 independent experiments
were conducted. These experiments confirm that the TRPM8 protein reconsti-
tuted into artificial lipidic bilayer resembles the properties of the native channel
and can be successfully used for studies.
Figure 6. A. Representative current/voltage relationship of TRPM8: Channels were incorporated in planar

lipid bilayers of synthetic POPC, POPE (3:1) in the presence of diC16 PtdIns(4,5)P2. Experimental conditions
are the same as described in the legend to Fig. 5. TRPM8 channels were stimulated with the application of
500 µM of menthol. The dashed line corresponds to the mean conductance of fully open channels, working
in inward direction, this state is rarely observed due to the low open probability of this subconductance level.
B: Representative current traces and all points’ histograms of outward (upper) and inward (lower) currents of
TRPM8 channels with clamping potentials were +60 mV and −60 mV, respectively. Experimental conditions
are the same as in the legend to figure 6A. C: Open probability of TRPM8 channels operating in inward and
outward directions measured at +100 mV and −100 mV. Data were analyzed from a total of 9 experiments. D:
Menthol dose response of the open probability of TRPM8. Demonstrated Po values were obtained at 100 mV.
Data were analyzed from a total of 36 experimens.
Figure 7. Activation of TRPM8 channels in Planar Lipid Bilayers by cold. Representative current traces
of TRPM8 activated by lowering the temperature from 23 to 16°C in planar lipid bilayers: Channels were
incorporated in planar lipid bilayers of synthetic POPC, POPE (3:1) in presence of diC16 PtdIns(4,5)P2.
Experimental conditions are the same as described in the legend to Fig. 6. Channels were inserted cis at 23°C
and the temperature was then lowered to 16°C at ~1 degree per min. Upper trace: TRPM8 activity at 23°C;
lower trace: TRPM8 channel activity at 16°C (representative of 12 independent experiments). The temperature
of the chambers was controlled by pyroelectric controller (see Experimental Procedures). The temperature in the
cis bath (ground) was read directly using a thermoelectric junction thermometer, which also served as a point of
reference for the pyroelectric controller. Data were filtered at 50 Hz. Clamping potential was −60 mV.
Further, we examined the effect of scPPX1 on the single channel activity of

TRPM8 in planar lipid bilayers (Fig. 8, 9). Channels were obtained in presence
of 1% diC16 PtdIns(4,5)P2 and 500 µM menthol with subsequent addition of
2 µg/ml scPPX1. No change in channel activity was detected when scPPX1 was
added to the external side of TRPM8. Conversely, addition of scPPX1 to the
internal side resulted in the inhibition of TRPM8 currents by affecting both the
open probability and conductance of the channel. We first analyzed the changes
in the open probability of the channel upon the cleavage of polyP. We found
that TRPM8 channel openings in inward direction were eliminated very rapidly
(within 1–2 min) after the addition of scPPX1. In contrast, the open probability
of the channel in outward direction exhibited much slower changes (up to 30
min). Figure 8A demonstrates current traces recordings obtained at −150 +150
mV voltage ramps before and after the treatment with scPPX1 in a time course
at the beginning of 3rd, 10th, 18th, 28th and 33rd minutes. The statistics of
the changes in open probability was obtained at different voltages in gap-free re-
cordings for TRPM8 alone or after the treatment with scPPX1 for the following
intervals of time: 5–7 min, 9–11 min, 14–16 min, 20–23 min, and 28–32 min
(Fig. 8B). Overall 16 experiments were conducted and open probabilities values
obtained for each voltage were derived from the analysis of at least 550–2636
events.
Figure 8. Voltage-dependence of TRPM8 before and after the treatment with scPPX1. A: Representative current
traces recordings obtained at −150 +150 mV voltage ramps before and after the treatment with polyphosphatase
in a time course at the beginning of 3rd, 10th, 18th, 28th and 33rd minutes. B: The changes in open probability
obtained at different voltages in gap free recordings for TRPM8 alone () or after the treatment with scPPX1
for the following intervals of time: 5–7 min (♦), 9–11 min (Δ), 14–16 min (), 20–23 min (◊), and 28–32
min (•). Data were analyzed from overall of 16 experiments.
Figure 9. Reduction of TRPM8 channel conductance by exopolyphosphatase scPPX1. A: Representative single-

channel current recordings of TRPM8 channels: upper traces – TRPM8 channels recordings before treatment
with scPPX1; middle traces – TRPM8 channel recording 15 minutes later after the addition of scPPX1 (2 µg);
lower traces – TRPM8 channel recordings after 30 minutes of addition of scPPX1. Clamping potential was
+100 mV. Data were filtered at 50 Hz. B: Symbols () (n = 5) correspond to the mean conductance values of
scPPX1 treated TRPM8 channels, where 2 µM of scPPX1 were added to the internal side of the channel; (ο)
(n = 8) mean conductance of control, untreated channels. Experimental conditions are the same as described in
the legend to Fig. 6.
Next we analyzed the effect of scPPX1 on channel conductance. We found

that, while TRPM8 inward currents were quickly eliminated by scPPX1, outward
currents were gradually inhibited within 30 min (Fig. 9). The upper traces of rep-
resentative currents and all points histogram in Figure 9A show TRPM8 activity
before the application of scPPX1; the middle traces and histogram were obtained
after 15 min of treatment with the enzyme; and lower traces and histogram show
the channel activity after 30 min of scPPX1 addition. The reduction of TRPM8
channel conductance is demonstrated in Figure 9B (n = 5). The single-channel
conductance approached zero upon cleavage of polyP with scPPX1. Channels not
treated with scPPX1 displayed no change in conductance within the time of the
experiments (n = 8).
Discussion
The activity of TRPM8 channels is regulated by a plethora of factors includ-
ing temperature, ligand binding, voltage, pH, etc. reflecting diverse stimuli and
mechanisms of these regulators. In our study, we found that inorganic polyphos-
phate plays an essential role in determining the activity of TRPM8 channels.
Furthermore, our data reveal that polyP is associated with TRPM8 in a supra-
molecular complex.
Inorganic polyphosphate is present in all eukaryotic cells, however its func-

tion is not well defined [25], [26], [27]. Despite the abundance of polyP in all
mammalian tissues, its evolutionary role and participation in many general physi-
ological processes in the cell, polyphosphate is still a mysterious molecule, since
little is known about the mammalian enzymes that control the levels of the poly-
mer. A novel mammalian exopolyphosphatase has recently been identified: the
DHH superfamily human protein h-prune, which shows high sequence homol-
ogy to the known PPXs, was shown to be an efficient exopolyphosphatase [43].
However, unlike other PPXs, h-prune hydrolyses only short-chain polyP, but the
long-chain polymers rather inhibit the activity of the enzyme. This leaves an open
niche for the existence of another enzyme that would cleave long-chain polyP. On
the other hand, endopolyphosphatase (PPN) activity was earlier found in several
mammalian tissues by A. Kornberg’s group [26]. The PPN activity was present
in rat tissues, particularly in brain, heart, lung and kidney, but the attempts to
identify the protein responsible for this activity have not been successful. Even
less is known about mammalian polyP synthases. No mammalian homologs to
the known polyP kinases (PPK) have been found in protein databases, and no
enzymes comparable to PPK have been identified. Also, it was reported that in
mammalian cells and tissues the synthesis of polyP from Pi bypasses the intracel-
lular pools of Pi and ATP [27], suggesting that the synthesis of polyP in animals
proceeds through a completely distinct enzymatic pathway, compared to one in
bacteria and lower eukaryotes.
PolyP – a molecule of many functions – has frequently been shown as an ac-
tive compound of several ion transporting systems, including some bacterial ion
channels [33], [44], [45]. Recently, it has been reported by Kim and Cavanaugh
that the presence of the polyanion is required for the regulation of the chan-
nel activity of one member of the mammalian TRP superfamily, TRPA1 [23].
The authors demonstrated using inside-out patches of HEK-293 cells expressing
TRPA1 that short polyphosphates (2–65 residues) are required for sensitizing this
channel to pungent chemicals and for preserving the protein in the functional
conformation. The activity of TRPA1 channels was recovered in excised patches
only in the presence of polyP and was diminished upon washing of polyP from
the bath solution, which suggests weak interactions between polyP and TRPA1.
Our own data show that polyP is also an important factor controlling channel
activity of TRPM8, however the nature of these interactions is quite different
from those observed by Kim and Cavanaugh. We found that polyP is co-purified
with the TRPM8 protein in ensemble and removal of polyP from the protein can
be achieved by its enzymatic digestion with scPPX1, indicating strong interaction
between TRPM8 and polyP.
We first found the requirement of polyP for TRPM8 channel activity in whole-
cell patch clamping of HEK-293 cells expressing TRPM8, where menthol-induced
currents were inhibited upon hydrolysis of polyP by scPPX1. Polyphosphatase
dialyzed via the patch pipette inhibited menthol-induced TRPM8 currents at −60
mV by 60% (Fig. 1A–C), while 80–90% inhibition was observed when scPPX1
was co-expressed with TRPM8 in HEK-293 cells (Fig. 1D–F, 2A–C). Enhanced
inhibition of TRPM8 currents by scPPX1 in these experiments suggests that a
longer exposure of the TRPM8 protein by expression of the enzyme ensures better
access to polyP than in those experiments performed with acute scPPX1 treat-
ment of TRPM8 via the patch pipette. The current/voltage relationship obtained
in whole-cell patch clamp experiments revealed that inward TRPM8 currents ex-
hibit a profound inhibition in the cells with co-expressed scPPX1 (~83%), while
outward currents are inhibited to a lesser extend (~65%) (Fig. 1G–I).
Alternatively, we monitored calcium signals in F-11 neuronal cells express-
ing TRPM8 with/out scPPX1 (Fig. 2D–F). Similarly to HEK cells expression
system, we found that Ca2+-entry was significantly inhibited in F-11 cells when
TRPM8 was co-expressed together with polyphosphatase, which indicates that
polyP plays an analogously important role on the TRPM8 activity in these cells
as well. These results demonstrate a novel and significant contribution of polyP to
TRPM8 channel activity.
The question of how polyP contributes to the regulation of TRPM8 chan-
nel activity motivated us to look at the biochemical and biophysical properties
of TRPM8 in a reconstituted system. In experiments on TRPM8 purified from
HEK-293 cells, we detected that polyP is associated with the protein (Fig. 4A).
The assembly of polyphosphate with bacterial membrane proteins has been previ-
ously reported for a number of ion channels, where it is usually derived in a com-
plex with the solvating polyester PHB [33], [44], [45]. PHB, possessing electron-
donating oxygens closely spaced along a flexible backbone, is capable of solvating
salts of hard cations; its amphiphilic nature allows it to penetrate hydrophobic
regions inaccessible to water. Considering possible interactions of TRPM8 with
the polymer, we further analyzed the protein for its association with PHB and
indeed found that TRPM8 is associated with the polyester (Fig. 4B).
Addressing the question of the nature of interaction of the two polymers with
the protein, we suggest that polyP possibly interacts with TRPM8 oligomers by
ionic bonds as it is found in the association with TRPM8 tetramers. However,
highly water soluble polyP might easily dissociate from the protein when it is
converted to the monomeric form. PHB, in contrast to polyP, is insoluble in
water and it is probably located in a hydrophobic region of TRPM8. Due to the
strong interactions with the protein, which might include hydrophobic or even
covalent bonds, PHB, unlike polyP, does not dissociate from the monomers of
TRPM8 (Fig. 4B, lane 2). Complexes of polyP/PHB have been shown to form
cation-selective channels in bacterial and mitochondrial membranes [30], [31],
[32]. Moreover, these complexes have been identified in association with some ion
channels including porins [33], [46].
An exciting model within these protein/polyP/PHB complexes has been rep-
resented by a potassium channel of Streptomyces lividans KcsA [44], [47]. As-
sociation of the polymers appears to contribute to the ion selectivity and gating
properties of KcsA, and to determine its preference between mono- and divalent
cations [34], [48].
It is conceivable that, in the case of the TRPM8 channel, similar formation
of a polyP/PHB complex might take place. Alteration of the polymers associated
with the protein could be a useful approach to demonstrate their function in the
complex. However, due to the amphiphilic nature of PHB it is difficult to elimi-
nate it from the native protein. For this reason, we concentrated our attention on
the soluble component of the complex – polyP – and its enzymatic degradation
by scPPX1.
In order to study the details of polyP participation in regulation of TRPM8
activity, we examined how scPPX1 modifies single channel activity of the purified
TRPM8 reconstituted into planar lipid bilayer. Although, one distant homolog of
TRPs, the polycystin-2 protein, that belongs to TRPP subfamily, has been shown
to form functional channels in lipid bilayers [49], no TRP channels from the
major subfamilies (TRPC, TRPV, TRPM) have previously been studied with this
technique, to our knowledge. We first aimed to identify the conditions necessary
to preserve the protein’s stability and function during extraction, purification and
subsequent incorporation into the artificial lipids. We found that it was critical to
deliver TRPM8 to the final step of purification in its tetrameric form, since alter-
ing different conditions, such as ionic strength, osmotic pressure or detergents
led to disintegration of tetramers into the lower assemblies, and ultimately to
monomers. The stability of TRPM8 in tetrameric form was also important to pre-
vent polyP dissociation. If such dissociation took place during purification, these
TRPM8 proteins failed to form functional channels. After finding the optimal
conditions for the purification, we attempted to detect TRPM8 channels after re-
constitution into planar lipid bilayers. By testing many experimental conditions,
we found that the TRPM8 channels in planar lipid bilayers can be activated by
both menthol and cold only in the presence of PtdIns(4,5)P2 (Fig. 5, 7). This
result was in agreement with previously reported data showing the requirement
of PtdIns(4,5)P2 for the channel activity of TRPM8 [11], [12]. To our knowl-
edge, our data provide the first demonstration of the dependence of mammalian
ion channel activity on PtdIns(4,5)P2 in a reconstituted system. This is strong
evidence that PtdIns(4,5)P2 regulates these channels through direct association,

and not through an intermediary PtdIns(4,5)P2 binding protein.
When the appropriate conditions were established, we tested the effect of scP-
PX1 on the channel activity of TRPM8 and found that addition of the enzyme to
the internal side of the channel was followed by drastic inhibition of the channel
activity. This was based on a reduction in open probability and single-channel
conductance to the point of complete loss of conduction (Fig. 8, 9). We detected
that while the inward currents were eliminated very rapidly, the outward currents
exhibited changes within 30 min of treatment with scPPX1. The kinetics of polyP
cleavage by scPPX1 is dependent on the Mg2+ concentration, where Mg2+ is a
cofactor for the enzyme activity [50]. In our studies, during exposure to the en-
zyme and polyP hydrolysis (Fig. 9B), inhibition followed a sigmoid time course,
which may have resulted from both the Mg2+ concentration and accessibility of
polyP from the protein.
Apparent voltage-dependence of the accessibility of polyP was also observed.
PolyP was more vulnerable to scPPX1 when inside-positive voltages were first
applied to provide an electrical driving force to favor an extension of polyP out
from the protein on the cytoplasmic side. This important observation suggested
a possible location of polyP in the inner cavity of channel conducting path and
led us to look closely whether alternation of polyP from the channel has an effect
on voltage-dependence of TRPM8. The voltage-dependence of TRPM8 has pre-
viously been reported by others [51], [52]. In our experiments we found that at
certain conditions TRPM8 exhibits a slight voltage-dependence, which, however,
undergoes significant changes upon the hydrolysis of polyP by scPPX1 (Fig. 8).
These results demonstrate that polyP not only co-purifies with TRPM8 protein,
but also modifies its function, and is thus directly involved in the regulation of
TRPM8 channel activity.
The inhibition pattern, caused by scPPX1, suggests two distinct mechanisms:
one resulted in a rapid decrease in channel openings observed in, physiologically
relevant to TRPM8, inward currents, while the other is seen as a slow decline in
conductance and channel openings detected in outward currents (Fig. 8, 9). The
molecular mechanism of this regulation is elusive at this time and more studies
are required for better understanding of the role of polyP in this channel/polymer
complex. One possible role for polyP in the activity of TRPM8 might be to de-
termine permeation and selectivity of the channel. Such a role of polyP has been
shown for a potassium ion channel KcsA [34], [48], [53]. It is also not clear how
scPPX1 treatment causes the reduction of single-channel conductance that we ob-
serve for TRPM8. It could be due to structural changes that may take place within
the intracellular domains of TRPM8 upon removal of polyP. On the other hand,
the reduced apparent single channel conductance could also be due to filtering of
rapid, incompletely resolved gating transitions, or a “fast block” process, due to

the uncovering of low affinity blocking sites of solutes by the removal of polyP.
In the context of the allosteric stimulatory and regulatory mechanisms that
affect TRPM8, our study demonstrates that the TRPM8 protein coexists in a su-
pramolecular complex with polyP and PHB, which alter its channel properties.
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On the Origin of Life
in the Zinc World:
1. Photosynthesizing,
Porous Edifices Built of
Hydrothermally Precipitated
Zinc Sulfide as Cradles
of Life on Earth
A. Y. Mulkidjanian
Abstract
Background
The complexity of the problem of the origin of life has spawned a large
number of possible evolutionary scenarios. Their number, however, can be
On the Origin of Life in the Zinc World 37
dramatically reduced by the simultaneous consideration of various bioener-

getic, physical, and geological constraints.
Results
This work puts forward an evolutionary scenario that satisfies the known con-
straints by proposing that life on Earth emerged, powered by UV-rich solar ra-
diation, at photosynthetically active porous edifices made of precipitated zinc
sulfide (ZnS) similar to those found around modern deep-sea hydrothermal
vents. Under the high pressure of the primeval, carbon dioxide-dominated
atmosphere ZnS could precipitate at the surface of the first continents, with-
in reach of solar light. It is suggested that the ZnS surfaces (1) used the solar
radiation to drive carbon dioxide reduction, yielding the building blocks for
the first biopolymers, (2) served as templates for the synthesis of longer biopo-
lymers from simpler building blocks, and (3) prevented the first biopolymers
from photo-dissociation, by absorbing from them the excess radiation. In ad-
dition, the UV light may have favoured the selective enrichment of photo-
stable, RNA-like polymers. Falsification tests of this hypothesis are described
in the accompanying article (A.Y. Mulkidjanian, M.Y. Galperin, Biology
Direct 2009, 4:27).
Conclusion
The suggested “Zn world” scenario identifies the geological conditions under
which photosynthesizing ZnS edifices of hydrothermal origin could emerge
and persist on primordial Earth, includes a mechanism of the transient stor-
age and utilization of solar light for the production of diverse organic com-
pounds, and identifies the driving forces and selective factors that could have
promoted the transition from the first simple, photostable polymers to more
complex living organisms.
Background
The problem of the origin of life is central to biology. It has been repeatedly ad-
dressed by scholars, including the above-quoted Erasmus Darwin and his famous
grandson Charles, who wrote in his letter to J.D. Hooker of February 1, 1871:
“It is often said that all the conditions for the first production of a living organ-
ism are now present, which could ever have been present. But if (and oh! what
a big if!) we could conceive in some warm little pond, with all sorts of ammonia
and phosphoric salts, light, heat, electricity, &c., present, that a proteine [sic]
compound was chemically formed ready to undergo still more complex changes,
at the present day such matter would be instantly devoured or absorbed, which
would not have been the case before living creatures were formed” [2]. Fifty years
later, Oparin has suggested, in the first comprehensive scenario of the abiogenic
origin of life (abiogenesis), that the primordial reducing atmosphere could have
favoured the spontaneous formation of proteinaceous bodies that could aggregate
into coacervates (protocells) [3,4]. Independently, Haldane, building upon the
achievements of virology, proposed that the life started from bacteriophage-like
molecules synthesized under the influence of the Sun’s radiation in the primordial
“hot dilute soup” [5]. It has been repeatedly demonstrated [6-17] that simple
building blocks such as amino acids or nucleobases could form from simpler com-
pounds, provided that energy was delivered as UV light or electric discharges (see
[18-36] for surveys of research on the origin of life, and a section below devoted
to the more detailed consideration of particular concepts).
The initial, rather general Oparin-Haldane’s concept of abiogenesis has been
gradually replaced by a mosaic of specific hypotheses that either emphasize the
“replication first” principle or build upon the “metabolism first” assumption. The
“replication first” concept implies that the emergence of the first replicating enti-
ties (replicators) preceded metabolism; it is represented by the RNA World sce-
nario that implies that RNA-like molecules capable of both self-reproduction and
simple metabolism were the first inhabitants of Earth [37-73]. The “metabolism
first” idea suggests that life started as a system of interacting chemical cycles and
the first replicators appeared later, see refs. [23,27,29,30,36,74-84] for consider-
ation of the controversy between the two concepts. Elsewhere, we have argued
that the “replication first” and “metabolism first” concepts complement rather
than contradict each other and have suggested that life on Earth started with a
“metabolism-driven replication” [85]. We have also emphasized that the virtually
unlimited number of tentative scenarios of the origin of life can be dramatically
reduced by the simultaneous consideration of a variety of external constraints
(boundary conditions) [85].
Here I invoke further (bio)energetic, physical, and geological constraints that
are related to abiogenesis. As a solution that satisfies these constraints, I put for-
ward an evolutionary scenario in which life on Earth emerged, powered by solar
irradiation, within porous edifices of hydrothermal origin that were built of pho-
tosynthesizing zinc sulfide (ZnS) crystals, in the “Zn world”.
Energetic, Physical, and Geological Constraints on

Abiogenesis
Energetics: Requirement for Utilizable Energy Flow(s)
Living organisms can exist only when supported by energy flow [86-92]. Be-
cause of the obvious requirement for energetic continuity, the energy flows that
deserve attention in an evolutionary context are those that remain constant on the
evolutionary relevant, geological timescale. This consideration discounts the evo-
lutionary importance of occasional energy inputs such as impact bombardment,
atmospheric electric discharges, and shock waves. The primordial atmosphere on
Earth is assumed to be dominated by carbon dioxide [25,93-100]. Hence, energy
was initially needed to reduce CO2 to organic compounds that could participate
in prebiological syntheses [34]. Currently, the fixation of CO2 by living organ-
isms is supported by two energy fluxes: the communities at the Earth’s surface
depend, via photosynthesis and its products, on solar light [101], whereas the
biotopes at the sea floor can also exploit the redox potential difference between
the reduced hydrothermal fluids and oxygenated ocean waters [102]. Accordingly,
some scholars have considered solar radiation to be the driving force of abiogen-
esis [5,85,103-112]. Others have hypothesized that chemical or redox disequi-
libria at the sea-floor hydrothermal vents [113-123] or at the surface of sea-floor
iron minerals [124-130] could have driven the emergence of the first organisms.
As argued in more detail elsewhere [85], a direct analogy between primordial life
and modern deep-sea biotopes is not possible, since the redox energy span of > 1
eV between the reduced compounds of hydrothermal fluids and the sea-dissolved
oxygen became exploitable only after the ocean waters – only 2 Ga ago – were sat-
urated by oxygen, a by-product of cyanobacterial photosynthesis [101,131,132].
The very lack of oxygen in the primordial atmosphere should, however, fa-
vour light-driven chemical syntheses. Without the ozone shield, the solar light
reaching Earth contained a UV component that was 10–1000 times stronger
than it is today [133,134] and could have driven diverse chemical reactions, in
particular carbon fixation. The major constituents of the primordial atmosphere
(CO2, N2, CH4, and water vapour [25,93-100]) let UV rays with λ > 240 nm
through [133]. The fossils of phototropic communities, which apparently flour-
ished as far back as 3.4–3.5 Ga [25,135-139], also indicate that the primordial
atmosphere was transparent to solar light. Hence, no other known energy source
could compete with solar irradiation in terms of strength and access to the whole
of the Earth’s surface.
Mauzerall has introduced an important additional constraint by noting that
the energy requirements of the first living beings had to be compatible with those
of modern organisms [109]. He argues that “the ur-cell would be simpler, but it
would also be less efficient”. More rigorously speaking, the intensity of the energy
flux(es) that supported the emergence of life should be either comparable with
the intensity of modern life-supporting energy flows or stronger. At least two
UV-driven abiogenic processes of CO2 reduction are known to proceed with
an efficiency comparable to that of modern photosynthesis. On the one hand,
the photo-oxidation of ferrous iron ions in solution can lead to the reduction of
CO2 [10]; for example, Borowska and Mauzerall have observed a light-driven
formaldehyde formation in the presence of dissolved ferrous hydroxide with a
quantum yield of 2–3% [140]. On other hand, a UV-driven synthesis of diverse
organic compounds from CO2 has been demonstrated at the surface of broad-
band semiconductors [141-149]. Such semiconductors not only photoreduce
CO2 but, depending on the initial substrates, can also photocatalyse a wide set
of diverse organic reactions [144,150-152]. Several naturally occurring minerals,
in particular TiO2 (anatase/rutile), WO3 (wolframite), MnS (alabandite), and
ZnS (wurtzite, sphalerite), possess the properties of broad-band semiconductors
and can photoreduce CO2 [107,143,151,153-158]. The highest quantum yield
of 80% has so far been reported for CO2 reduction to formate at the surface of
colloidal ZnS particles [144,145].
Physics: Photostability of Nucleotides

RNA and DNA are polymers of similar sugar-phosphate units, with each sugar
moiety (ribose in RNA or deoxyribose in DNA) carrying one of four different ni-
trogen bases (nucleobases). The specific feature that is shared by all nucleobases is
their unique photostability [159-165]. Since this trait is not related to the storage
of genetic information, several authors [105,112,133,159,164,165] have noted
that this property could have been of some use when the UV flux at the surface
of primordial Earth was much stronger than it is now [133,134]. Nucleobases
apparently can absorb excess energy quanta from sugar-phosphate moieties and
protect them from photo-dissociation [166]. This feature explains why the UV
damage to the backbones even of modern RNA and DNA molecules is 103–104
times less frequent than destruction of nucleobases themselves [159].
Based on a Monte-Carlo simulation of primordial photochemistry [112], we
have proposed an evolutionary scenario in which the relative enrichment in in-
creasingly complex RNA-like polymers could be attributed to their higher pho-
tostability in a UV-irradiated environment, with UV-quenching nucleobases pro-
tecting the sugar-phosphate backbones from photo-dissociation. It was posited
that the photostability could increase further owing to the stacking of nucleobases
and the formation of Watson-Crick pairs [85,112], see also below.
In modern organisms, the continuous victimization of nucleobases is a well-
known problem that is counteracted by sophisticated repair systems [167]. At the
earliest steps of evolution, repair systems were absent, so the photodestruction of
nucleobases could have hindered the selection of the first replicators. The photo-
destruction of nucleobases could be, however, prevented by radiation-absorbing
templates. Many minerals can take up radiation energy from the adsorbed pho-
toactive compounds. For example, montmorillonite particles have been shown to
protect catalytic RNA molecules (hairpin ribozyme 1) from UV-induced damage:
after a UV-irradiation, the self-cleavage activity of the montmorillonite-adsorbed

ribozyme molecules was three times higher compared to that of the molecules irra-
diated in the absence of montmorillonite [168]. With ZnS crystals, the excitation
transfer from adsorbed dye molecules to a template has been shown to proceed
within picoseconds [169], i.e. much faster than the typical intrinsic characteris-
tic time of photodestruction (e.g. ~20 μs for adenosine monophosphate [170]).
Hence, in the evolutionary context, the first photostable RNA-like polymers had
better survival chances at the surfaces of those minerals that could efficiently ab-
sorb the radiation energy.
Geology: Requirement for Hydrothermal Settings

In addition to abundant chemical elements such as carbon, oxygen, nitrogen, and
hydrogen, biological systems contain a number of microelements, often at levels
far exceeding those in the surrounding environment (see [171] for a comprehen-
sive survey). In particular, transition metals are often involved in enzyme catalytic
centres [90,172]. The concentration of such metals in modern cells is many or-
ders of magnitude larger than that in sea water (see Table 1); the ion accumula-
tion is accomplished by sophisticated transport systems and demands ion-tight
membranes to prevent the escape of trapped metal ions out of the cell [173,174].
However, the ion-tight membranes, as argued elsewhere [175,176], seem to be
a relatively late evolutionary acquisition. Here we encounter a paradox. On the
one hand, the emergence of metal-containing RNA and protein domains – as a
result of their eventual stabilization by available transition metal ions – implies
an abundance of these ions. On the other hand, the equilibrium concentration of
such ions in sea water is very low (see Table 1). This paradox is routinely resolved
by invoking hydrothermal settings as potential cradles of life [113-123,177]. In
Table 1. Approximate total concentration of key ions in various environments (in moles/litre).
such systems, which currently cluster around the mid-ocean ridges and deep-sea
submerged volcanoes (seamounts) – where hot magma chambers occur near the
seabed – water circulates down into the crust, becomes heated, and then rises up.
When water is overheated to more than 400°C, it can leach metal ions from the
crust. These ions are then brought to the surface by hot hydrothermal fluids, so
that the steady-state concentrations of metal ions at the orifices of hydrothermal
vents may exceed the equilibrium concentrations because of this continuous sup-
ply [102,178].
Since hydrothermal fluids are rich in H2S, the interaction of metal-rich hot
hydrothermal fluids with cold ocean water leads to the precipitation of metal
sulfide particles that form “smoke” over the “chimneys” of deep-sea hydrothermal
vents [102,178]. These particles eventually aggregate, settle down, and, ultimate-
ly, form porous, sponge-like structures around the vent orifices [179-181]. The
vent systems have a zonal structure [102,178,182]: pyrite (FeS2) and chalcopyrite
(CuFeS2) are found in the centre, where the temperature of hydrothermal fluids
is the highest (~350°C; the water at the sea floor remains liquid even at such high
temperatures because the pressure is above 200 bar [102]). At the periphery of
hydrothermal fields, the temperature of hydrothermal fluids is lower because the
rising hot fluids mix, while still under the sea floor, with the cold ocean waters
that are pressed into the seabed by the overlying water column. Those peripheral
chimneys that eject fluids with temperature in the range of 200°C to 300°C are
covered by porous precipitates of sphalerite (ZnS), with additions of other sul-
fides such as galena (PbS) and alabandite (MnS) [102,180,182]. This change in
chemical composition is due to the fact that upon cooling, the sulfides of iron and
copper precipitate much faster than those of zinc and manganese [183]. Accord-
ingly, when the temperature of hydrothermal fluid is less than 300°C, the sulfides
of iron and copper precipitate already below the sea floor, inside the moulds of
hydrothermal systems, so that the transition metal ions that reach the surface are
predominantly Zn2+ [184]. The difference in the precipitation rates manifests it-
self also in the chemical composition of those vent chimneys that eject both Fe2+
and Zn2+ ions. The throats of such chimneys are formed of promptly precipitat-
ing FeS and CuFeS2, while their outer surfaces are coated by the more slowly
precipitating ZnS [179,181].
The zonal structure is remarkably conserved between the modern hydrother-
mal vent systems and the ancient volcanogenic massive sulfide (VMS) deposits
of hydrothermal origin. VMS deposits can reach many kilometres in diameter,
and date back to the Archean period [185-187]. The ancient VMS deposits have
pyrite (FeS2) and chalcopyrite (CuFeS2) at their centres being encircled by con-
secutive halos of, e.g., pyrite-chalcopyrite-sphalerite, sphalerite-galena-alabandite,
and, finally, chert [185,187]. ZnS crystals: Unique traits
The above listed constraints, when considered simultaneously, identify crystal-

line ZnS as the single compound that (i) can serve as an efficient photocatalyst
capable of reducing CO2 with a quantum yield of up to 80%, (ii) can promptly
absorb UV quanta from the adsorbed organic compounds, preventing their de-
struction, and (iii) is a major constituent of hydrothermal vent systems, being
typically found at their outer surface and/or periphery.
The evolutionary scenario that is given below suggests that porous ZnS forma-
tions of hydrothermal/volcanic origin performed several functions, being involved
in the primeval photosynthesis of the first metabolites, in the (photo)selection of
the first RNA-like polymers, and in their protection from photodestruction.
Hypothesis: Emergence of the First Biopolymers

at Photosynthesizing ZnS Edifices of
Hydrothermal Origin
Initial Geological Settings
After the primary hydrogen atmosphere of Earth had escaped into space, the so-
called secondary atmosphere built up with volcanic gases; this atmosphere was,
most likely, dominated by CO2, with smaller amounts of N2, CO, and H2, similar
to that on modern Mars and Venus, where CO2 still makes up 95% of the atmo-
sphere [25,93-100]. As the Earth’s surface gradually cooled, water vapour started
to condense into the first oceans. The atmospheric pressure at the surface of pri-
mordial Earth has been estimated to reach several hundred bars; therefore ocean
formation could have started when the surface was still very hot [94,97,100].
Zircon data indicate the presence of the first continent(s) by 4.2 Ga [188]. Hy-
drothermal activity of some type would have been established promptly, driven
by thermal convection. Most likely, the initial convection systems did not form
a continuous chain of mid-ocean ridges as they do now but a pattern of “hot
spots” similar to modern volcanic island arcs [93,95,189]. The activity of these
hydrothermal/volcanic systems was accompanied by surges of hot hydrothermal
fluids to the surface. Owing to the initial atmospheric pressure of ≥ 100 bar (see
[94,100], and cf. with the pressure of 95 bar at the surface of modern Venus), very
hot hydrothermal fluids enriched by dissolved metals could discharge directly to
the surface of the first continents. This situation differed fundamentally from
the modern one, since today such hot fluids can reach the continental surface
only as steam (at the points of volcanic or geyser activity), losing their metal
content on the way. Taking into account the slow precipitation of ZnS under
high-pressure conditions [183] and the abundance of Zn in the Earth’s crust [90],
one can expect a major delivery of Zn-enriched hot fluids to the surface of the
first continent(s). Zinc could even have been the dominant transition metal in the
continental hydrothermal fluids when the atmospheric pressure changed in the
range from ca. 100 bar to ca. 10 bar (this would correspond to the temperature
of hot fluids reaching 300°C – 200°C, respectively, cf. with the above described
situation at modern hydrothermal vents). Hence, it is possible that the large ar-
eas of first continent(s) could have been covered by porous ZnS precipitates of
hydrothermal origin. These ZnS edifices should have been accessible to solar UV
radiation at the surface of continents and in shallow waters surrounding them
(see Fig. 1). Hereafter, the term “sub-aerial” is used to denote illuminated settings
where the UV-rich solar light could have served as an energy source for primordial
syntheses (see also [25,114,177]).
Figure 1. Primeval ZnS-mediated photosynthesis in sub-aerial, illuminated settings. Right: Precipitation of

FeS and ZnS nanoparticles (black and grey spots, respectively) around a primeval, sub-aerial hot spring. Note
that ZnS and FeS particles precipitate at different distances from the spring. The picture is based upon data
from [102,119,179,181,183]; see the main text for further details. Left: A schematic presentation of reactions
within a photosynthesizing ZnS nanoparticle, as combined with an energy diagram; the scheme is based on
refs. [145,149,190,278]. Initially the absorption of a UV quantum leads to the separation of electric charges.
The electrons migrate in the crystal until they are trapped at the surface; the trapped electrons can reduce a
CO2 molecule either via two one-electron transfers [144] or, possibly, in a concerted two-electron reaction. The
electron vacancy (hole) is initially reduced by the S2- ion of the crystal; the ultimate electron equilibration, as
discussed in the main text, requires external electron donors, e.g. H2S. Note that, for simplicity of presentation,
the one-electron and two-electron reactions are not discerned; see the main text for further details.
ZnS-Mediated Photosynthesis
In the absence of an ozone layer, the UV component of solar radiation would
have driven the reduction of CO2 at the ZnS-covered surfaces. Since these
surfaces were formed by precipitated ZnS particles (see Fig. 1), similar to those used
in the aforementioned experiments with the photosynthetically active “colloidal”
ZnS crystals [144,145], the reduction of CO2 may have proceeded with a high
quantum yield under the high atmospheric CO2 pressure. Zinc sulfide is a very
powerful photocatalyst that, besides reducing CO2, is capable of driving diverse
reactions of carbon- and nitrogen-containing substrates [144,150,152,190-192].
Such substrates could build up in the atmosphere, be generated by photochemi-
cal reactions in the water phase [24,106,109], accompany volcanic extrusions
[193] and hydrothermal fluids [123], or be brought by meteorites [194]. They
could have then participated in further photocatalyzed transformations at the
ZnS surfaces.
First Settlers in the ZnS World

Electrically charged products of photosynthesis, e.g. negatively charged carbonic
acids, would have been attracted by the complementary charges at the ZnS sur-
faces of mineral compartments. These molecules could have interacted with each
other at the catalytic surfaces of continuously operating porous ZnS photore-
actors, yielding even more intricate carbon- and nitrogen-containing molecules.
The more complex molecules would, generally, have absorbed more light and
been more vulnerable to UV quanta. Still, in certain cases, the increase in chemi-
cal complexity may have been accompanied by an increase in photostability. In-
deed, the destruction of a chemical compound by a light quantum starts with the
“trapping” of its energy by a particular chemical bond, followed by an increase
in the energy of this bond and its eventual dissociation [195]. However, if the
absorbed energy is spread over many bonds, then the probability of bond cleavage
drops dramatically. Such a spreading of excitation energy occurs in systems that
contain conjugated bonds (so-called π-systems with alternating single and double
bonds); the spreading is facilitated by a ring-like (aromatic) molecular structure.
All nucleobases belong to such ring-like, conjugated systems [159]; the lifetime of
their excited states (~100 fs [164]) is extremely short even for π-systems; this short
life time would additionally have decreased the probability of photodestruction.
As discussed above, the UV-resistance of π-systems could increase further upon
their stacking together and/or adsorption to radiation-absorbing minerals.
Hence, at illuminated ZnS surfaces, the UV-resistant, ring-like compounds
could survive as stacked aggregates of “rings joined to rings” (quoted from [1]). A
relative enrichment in such stacks, as well as their stabilization by covalent link-
ages, could be driven by a number of factors, namely, the UV-resistance of polym-
erized and stacked π-systems [112,164], the potential to utilize the energy of UV
quanta for photopolymerization [11,12,196], the ability of Zn2+ ions to catalyze
the polymerization [197,198], and the low dielectric permittivity of the surface-
adjoining water layers [199] that may have favoured condensation reactions. In
addition, a regular mesh of electric charges at the ZnS surface, by attracting re-
actants and arranging them appropriately relative to each other, may have made
the polymerization thermodynamically more favourable than in bulk water [124].
Then, however, the resulting polymers should have stayed confined to the surface
[200]. In the context of a UV-irradiated environment such confinement could be
considered a rescue since an eventual detachment of a primordial polymer from
the energy-absorbing ZnS surface would have led to faster photodestruction. At
the same time, while prevented from detachment from the surface, the molecules
would have been able to diffuse along the surface, to interact with each other,
and to form aggregates, which is a pre-condition for the abiogenic emergence of
increasingly complex structures.
The scenario outlined above should yield two fundamentally different popula-
tions of molecules, namely surface-confined, relatively complex structures capable
of efficient discarding excess radiation energy (hereafter referred to as zymes), and
continuously photosynthesized simpler organic molecules – the future metabo-
lites – that stored the solar energy in their covalent bonds.
First Replicating Entities

The eventual elongation of zymes would promote their entropy-driven folding
at the surface. In addition, an increase in their amount would encourage inter-
actions between them. Both factors may have led to the formation of hydrogen
bonds within zymes and/or between different zymes. Clustering of zymes may
have been favoured by high pressure [201] and periodic drying events, e.g. in tidal
regions [18,202], resulting in a kind of natural polymerase chain reaction (PCR)-
like process [203]. The UV-stability of double RNA strands, owing to hydrogen
bonding, is much higher than that of single-stranded RNAs [159,164,204-206].
Therefore those π-systems may have been selected – from the initially larger set of
compounds – which could make multiple hydrogen bonds with each other. Ulti-
mately, this selection would have led to a relative enrichment of complementary
nucleobases, including those that we know now. It is plausible that the π-systems
could have been initially linked in various ways. It would appear that one of these
constructs, the one with ribose-phosphate units connecting the stacked nucle-
obases, attained the ability for self-replication and was, because of this, retained
by evolution.
Unlike occasional self-replication events, a systematic, accurate replication
is thermodynamically demanding [207] and would require special machinery
that could consist of several folded polymers resembling modern transfer RNA
(tRNA) or ribosomal RNA (rRNA). It is unlikely that we would ever be able to
reconstruct all the steps that led to the formation of first replicators (see but refs. [
44,52,54,58,60,65,67,72,208,209] for tentative scenarios). We can, however, try
to make guesses on the selective forces underlying their emergence. At least two
factors deserve note:
1) Nucleobases quench the UV quanta by converting the energy into heat.

The heating, however, is not harmless. Using the approach of Dancshazy
and co-workers [210], it is possible to surmise that a single UV quan-
tum should locally heat a 100-unit RNA-like polymer by tens of degrees
Celsius. Even if the absorbed energy can be promptly transmitted to a
template, local heating of the template can eventually cause ablation of the
molecule [211]. In the UV-irradiated environment, both outcomes could
lead to polymer deterioration. Overheating, however, can be avoided by
channelling part of the energy into work. For example, those zymes that
could serve as antennas and use the UV energy, e.g., for connecting nucle-
otides together (as hypothesized by Skulachev [111]) had better survival
chances. This selective advantage of “working” polymers over the “idle”
ones is general in nature: it is applicable not only to the first ribozymes,
but also to the first proteins, as discussed in the next section.
2) A population of RNA-like polymers adsorbed at a ZnS template could
gather light and therefore enhance the yield of the photocatalysis. Since
the hydrothermal ZnS structures are highly porous [179-181], the pores/
compartments that contained efficient replicators and, hence, an increas-
ing number of UV-absorbing zymes could produce more metabolites,
which, in turn, could be used to build new replicators, resulting in a kind
of positive feedback.
As first pointed out by Horowitz [212], and as our simulations showed ([112];
see also the section on energetics of the Zn world below), an abiogenic formation
of complex molecules (e.g. long polymers) would imply the presence of an over-
whelmingly larger amount of simpler molecules of the same type (shorter poly-
mers). The first replicators could therefore utilize the available shorter fragments;
with time, they could acquire the ability to attain building blocks by cleaving the
(phosphor)ester bonds in other surface-adsorbed zymes. Owing to this develop-
ment: (i) a coupling mechanism could be established that, with some modifica-
tions, has been in use ever since – the polymerization of RNA and DNA is still
driven by cleavage of the phosphoester bonds in ATP; and (ii) the genuine fight
for survival could begin, since each replicator became a potential prey for others.
Emergence of Proteins and Enzymes

Generally, the thermodynamics of primordial syntheses of polymers, and in par-
ticular of polypeptides, has been deemed a riddle since the respective condensa-
tion reactions, which are accompanied by the release of water molecules, should
be unfavourable in water (see e.g. refs. [83,213] for further discussion of this
point). A closer inspection of modern metabolic chains offers a way out of this
conundrum. Although biological syntheses are indeed accompanied by the release
of water molecules, they never proceed “for free”, but are coupled to thermo-
dynamically favourable, exergonic reactions, e.g. of ATP hydrolysis, which re-
quire water. Hence, if (i) there were substrates that could be hydrolyzed and (ii) a
thermodynamic coupling between syntheses and hydrolyses could be established
– then the primordial syntheses could proceed without violating the laws of ther-
modynamics.
In this framework, the emergence of new synthetic pathways could proceed in
two steps, as follows. First, a (ribo)zyme capable of cleaving a new type of chemi-
cal bond – in a thermodynamically favourable reaction – would have emerged.
Then, the ability to make this particular type of chemical bond could develop as a
reversal of the new catalytic pathway, provided that coupling with some exergonic
reaction (e.g. a phosphate group transfer or a hydrolysis of a phosphoester bond)
could be established.
In the ZnS settings, photosynthetically produced polycarbonic compounds,
inorganic polyphosphates, RNA-like oligomers, and diverse low-molecular-weight
phosphorylated derivates could serve as cleavage substrates for the first synthases.
The reducing equivalents that would be needed for some synthetic reactions were
continuously generated at the illuminated ZnS surfaces; nucleotide-containing
redox cofactors, such as NADH, NADPH, FAD, and FMN [22,214-216], could
have emerged as mediators that picked up the photoexcited electrons from photo-
active ZnS surfaces and delivered them to the respective ribozymes.
After the emergence of the first replicators capable of connecting amino acids
by peptide bonds, the synthesis of first, random polypeptide sequences at nucle-
otide and/or ZnS templates could begin. Without considering the elusive chemical
details, it seems fitting, as in the previous section, to focus on selective factors that
could drive the emergence of the first polypeptides. They may have been recruited
to perform functions that did not require a particular amino acid sequence but
only the ability of a polypeptide to bind to a replicator, e.g. via the protein back-
bone groups [209]. Such a binding, for example, could protect the backbone of
replicators from hydrolysis or cleavage. In addition, the bound polypeptides could
absorb heat into which the energy of UV quanta was converted, therefore pro-
tecting the replicators from thermal damage. It is noteworthy that, if a protecting
polypeptide could eventually discard excess energy by funnelling it into a chemical
reaction, then the probability of denaturation would have become lower. Those
replicators that could synthesize proteins capable of supporting catalysis by (ribo)
zymes [67] or catalyzing useful chemical reactions themselves may have got an
advantage. Eventually such a selection could establish a correspondence between
the nucleotide and amino acid sequences, see [46,47,53,58,67,73,217,218] for
tentative scenarios on the evolution of genetic code and protein synthesis.
The First Colonization Wave

The ZnS precipitates would have attenuated the UV component of solar light,
thus providing shade for the inhabitants of the lower-lying compartments. Hence
a stratified system could be established with the illuminated upper layers account-
ing for maximal photosynthesis, and lower, less productive, but more protected
layers providing shelter for the first replicating entities in their pores. The poros-
ity of the ZnS precipitates [179,180] would have enabled metabolite transport
between the layers. Moreover, the sponge-like inner structure could eventually
enable variable hydration of the compartments that may have served as an ad-
ditional selective factor. Both the gradient of light and the interlayer metabolite
exchange are typical of modern stratified, seashore phototrophic communities
(e.g. stromatolites [25,93,139]).
Those consortia of replicators that were able to couple useful syntheses with
exergonic chemical reactions could have became relatively independent of upper,
photosynthesizing layers and could penetrate the depths of their porous habitats.
This penetration can be considered the first wave of the Earth’s colonization by
living organisms that eventually evolved from replicating zymes. This coloniza-
tion would have been supported by exchanges of metabolites between the illumi-
nated strata and those that were deeper and darker. In the darkness, direct contact
between the RNA-like “bodies” of replicators and the radiation-absorbing ZnS
template was no longer crucial, so that the replicators could evolve into life forms
that were enclosed in protein envelopes resembling modern viruses [62,70,219].
The energetics of these communities consisted in the interplay between the
continuous ZnS-mediated photosynthesis and the increasingly complex het-
erotrophy of the first organisms. This heterotrophy may have been based on cou-
pling the exergonic breakdown of photosynthetically produced metabolites with
endergonic (bio)synthetic reactions. This same interplay of photosynthesis and
heterotrophy still drives the majority of terrestrial communities today.
The Fall of the Zn World

Upon further decrease in the amount of CO2 in the atmosphere and, accordingly,
in atmospheric pressure, the delivery of Zn-rich fluids to the surface of continents
would have gradually ceased, so that fresh, photosynthetically active ZnS surfaces
could no longer form in sub-aerial settings. After that, ZnS-rich hydrothermal
edifices could persist only deeply at the sea floor, ultimately clustering around the
mid-ocean ridges. The organisms that remained confined to the sub-aerial settings
would have found alternative ways to reduce CO2. Moreover, in the absence of a
zinc supply, they were forced to confront unfamiliar minerals, in particular those
containing iron. At that time the dominating transition metal ion in sea water
would have been Fe2+ [90,220,221]. Iron, unlike zinc, is redox-active and can
generate harmful hydroxyl radicals [222-225]. It would seem that the expatriates
of the Zn world had to be full-fledged organisms with reliable replication machin-
ery, robust metabolism, and protective envelopes. The story of how they could
populate the Earth is beyond the problem of the origin of life proper and hence
remains out of the scope of this communication; this topic, however, is addressed
in the accompanying article [226].
Validation of the Hypothesis

The search for a solution to the origin of life puzzle is hindered by the impos-
sibility of providing an ultimate experimental proof, namely to (re)produce life
from scratch de novo (see e.g. [58,218]). In view of this obstacle, Wächtershäuser
[227] has suggested that the hypotheses on the origin of life could be validated
by rigorous application of Karl Popper’s principles of testing scientific theories
[228,229]. Popper wrote: “We may if we like distinguish four different lines along
which the testing of a theory could be carried out. First there is the logical comparison
of the conclusions among themselves, by which the internal consistency of the system
is tested. Secondly, there is the investigation of the logical form of the theory, with the
object of determining whether it has the character of an empirical or scientific theory,
or whether it is, for example, tautological. Thirdly, there is the comparison with other
theories, chiefly with the aim of determining whether the theory would constitute a
scientific advance should it survive our various tests. And finally, there is the testing
of the theory by way of empirical applications of the conclusions which can be derived
from it” (quoted from ref. [229]). The core of Popper’s approach is the idea of
falsification, i.e. putting a hypothesis to the test by making testable predictions
and checking them. The falsification tests of the Zn world hypothesis, because
of their key importance, are described in a separate accompanying article [226].
Here I consider only the empirical supporting evidence, the internal consistency
of the hypothesis, and the relation of the posited concept to other hypotheses on
the origin of life.
Supporting Evidence
Below, the supporting data are organized along the steps of the above-presented
evolutionary scenario. One outstanding feature, however, deserves preferential
treatment since it is crucial for almost every one of these steps. This feature is the
unique ability of ZnS crystals to store radiation energy (see [144,230-233] for
reviews). This property manifests itself in phosphorescence (afterglow), so that
ZnS – widely known as “phosphor” – is used in numerous devices, from various
types of displays to ‘glow in the dark’ items. ZnS is a broad-band n-type semi-
conductor with a gap band energy of 3.2–3.9 eV (depending on the particular
crystal structure). When radiation strikes such a semiconductor, it may excite an
electron and consequently leave a “hole” (see Fig. 1). If the energy of the radiation
quantum is larger than the band gap energy, the electron reaches the so-called
conduction zone and can move inside the crystal. Ultimately, the electron can
recombine with the hole. However, if the electron gets into an energy trap (see
Fig. 1), then the recombination can proceed only slowly, under the condition of
thermal activation [234]. In pure ZnS, the atoms at the surface can trap electrons
[234,235], particularly if the semiconductor interacts with a polar solvent such as
water. At the surface, a photoexcited electron loses part of its energy owing to the
interaction with the molecules of the solvent and, accordingly, is prevented from
returning into the bulk of ZnS. In addition, recombination may be prevented by
prompt replenishing the hole with an electron coming from a potent electron do-
nor (“hole scavenger”). The trapped electron then remains confined to the surface
until an external electron acceptor, e.g. a molecule of CO2, picks the electron up,
see [144,190,234,236] for reviews.
Small nanocrystals of ZnS and of its chemical “twin” CdS have been found to
behave as quantum dots – systems with physical properties intermediate between
those of bulk materials and single molecules [144,234,237-242]. Accordingly,
these nanoparticles have drawn much attention as promising fluorescent labels
for biology, so that the interaction of proteins and nucleic acids with the CdS
and ZnS quantum dots has been intensively studied (see [242-248] for reviews).
It is noteworthy that the ZnS particles obtained from hydrothermal vent plumes
could pass through 200 nm filters [249] and, hence, can be reasonably categorized
as nanoparticles.
Initial Geological Settings

An overall picture of primordial Earth, as based on available data, has been given
in Section 2.1. The question that is crucial for the Zn world scenario is whether
atmospheric pressure was in the range of 10–100 bar when life on Earth emerged
(at this pressure range, hot hydrothermal fluids surging at sub-aerial settings would
have been enriched in Zn; see above). It is generally accepted that life on Earth
was unlikely to emerge before the end of the impact bombardment 3.9 Ga ago
[25,26,80,93,95,96]. Since reliable biogenic fossils are dated to 3.4–3.5 Ga [135-
139], life is believed to have emerged from 3.9 to 3.5 Ga [25,26,34,80,93,95,96].
Concerning the primordial atmospheric pressure, the following estimates have
been put forward. Atmospheric pressure before condensation of the first ocean
ca. 4.4 Ga ago has been estimated as ≥200 bar; this ocean condensation would
have lowered the pressure to about 100 bar [94,100]. Accordingly, each evapora-
tion of ocean that may have been caused by massive bombardment from 4.3 to
3.9 Ga would have transiently increased the atmospheric pressure. Atmospheric
pressure 3.3 Ga ago has been estimated to have been in the range of 2–6 bar,
depending on the assumed concentration of methane in the atmosphere [97]. It
is remarkable that these estimates are compatible with the atmospheric pressure
values in the range from ca. 100 bar to ca. 10 bar in the time window from 3.9 to
3.5 Ga. Hence, the time window when the hydrothermal ZnS could precipitate
at sub-aerial settings overlaps with the time window when life is believed to have
emerged on Earth.
The conditions under which the first cells emerged could be read from the
chemical composition of cellular cytoplasm that apparently tends to maintain
the ancient chemical milieu [25,34,84,85]. Archaeal, bacterial, and eukaryotic
cells all show elevated concentrations of potassium, magnesium, phosphate, and
certain transition metal ions (see Table 1). The elevated amount of transition met-
als, as already noted, could be attributed to the emergence of life in hydrothermal
settings [25,93,113-115,177]. Not in contradiction with this attribution, the el-
evated amounts of K+ and Mg2+ might reflect the involvement of volcanic activi-
ties: elevated and positively correlated amounts of potassium and magnesium are
typical of certain volcanic rocks [250].
The high phosphate concentration in cells might indicate the abundance of
phosphorus compounds in the primordial waters [85,251,252]. Since the con-
centration of phosphate in sea water is low (the phosphates of Ca and Mg are
poorly soluble in water), it has been argued that more reduced compounds such as
hypophosphite (phosphinate, PO22-) and/or phosphite (phosphonate, PO33-),
which have better solubility in sea water, could have been abundant in the pri-
meval, more reduced ocean [252-258]. This suggestion is supported by findings
of diverse systems of hypophosphite and phosphite oxidation in prokaryotes (see
[259] for a review).
The high extent of hydrothermal Zn delivery to the surface of Earth during
the earliest stages of its history is reflected in the association of present day major
zinc ore deposits (built of ZnS) with Archean oceanic spreading centres and island
arc terrains [260,261].
In summary, these features point to environments similar to those in which

VMS deposits originated (see [187,262] for reviews). The largest VMS deposit
is believed to be the Iberian Pyrite Belt, which is 200 km long and 40 km wide.
The oldest VMS deposits discovered so far, dated 3.2–3.5 Ga, are at the Pilbara
Craton of West Australia [25,263,264]. Remarkably, besides its huge zinc content
(zinc lenses) [265,266], the Pilbara Craton also contains filamentous microfossils
[267,268]. It is tempting to speculate that the Pilbara Craton could serve as a
rough model of an environmental setting where life could have emerged earlier, at
the end of the Hadean, when ZnS could precipitate, because of high atmospheric
CO2 pressure, within reach of solar UV radiation.
Photosynthesis in the Zn World

The ZnS-mediated photosynthesis of diverse organic compounds from CO2, in
view of its potential practical application, has been studied intensively, starting
in the early 1980s [144-146,232,236,269-273]. Besides the reduction of CO2 to
formate (see Fig. 1 for a scheme), the photosynthesis of dicarbonic, tricarbonic,
and tetracarbonic acids has also been shown [146,155]. Yanagida and co-workers
have reported the photoreduction of diverse acyclic and cyclic ketones to the cor-
responding alcohols [274,275] and the ZnS mediated formation of diethylam-
ine from ethylamine [192]. It is noteworthy that the highest quantum yields of
CO2 photoreduction, in the range of 10–80%, were obtained, by various research
groups [144,145,148,149,155,273], with ZnS nanoparticles – analogues to those
ejected by hydrothermal vents. These nanoparticles are particularly photosynthet-
ically active since the probability of electron trapping at the surface increases with
the surface/volume ratio [241,276]. Studies of the photoelectrochemical reduc-
tion of CO2 at p-type semiconductors showed that at high CO2 pressure the rate
of reduction was principally limited only by light intensity [277].
While studying photochemical hydrogen production in suspensions of ZnS
particles (with quantum yields reaching 90%), Reber and Meier found that the
most stable, long-lasting hydrogen production was observed when a mixture of
sodium sulfide and sodium hypophosphite was used as a hole scavenger [278].
This system appears to be very interesting in the evolutionary context. Here the
accumulation of disulfide ions (S2 2-) that could be caused by sulfide (S2-) oxida-
tion by photogenerated hole (h+) via a sulfide anion radical (S·-) as an intermedi-
ate according to the equations
s 2− + h + → s ⋅− (1)
s ⋅− + s⋅− → s22− (2)
was prevented by the reductive action of hypophosphite that yielded phosphite,

so that sulfide was recycled:
s22− + H 2 PO2− + 3OH − → 2 S 2− + HPO3− + 2 H 2O (3)

After all the hypophosphite wass consumed, hydrogen formation proceeded fur-
ther, although slower, owing to the further oxidation of phosphite into phosphate
[278]:
S 22− + HPO3− + 3OH − → 2 S 2− + HPO43− + 2 H 2O (4)
The high efficiency of (hypo)phosphite as a hole scavenger was also demon-

strated by Kanemoto and co-workers, who studied, in addition to the photogene-
ration of hydrogen, the photoreduction of CO2 by ZnS [273]. As already noted,
hypophosphite and phosphite may have been abundant in the primordial ocean
[252-254,256-258] and could participate in the primeval photosynthesis. More-
over, phosphate ions, ultimate products of the (photo)oxidation, owing to their
affinity to metal sulfides [279], would have remained adsorbed at the ZnS surface,
providing a specific phosphate-rich reaction milieu.
The aforementioned photo-oxidation of sulfur aniones can eventually lead
to disruption of the ZnS crystals and release of Zn2+ ions that could not be
completely prevented even by efficient hole scavengers [271]. In the context of
primordial photochemistry, such a photocorrosion would have led to the continu-
ous rejuvenation of the ZnS surfaces and the formation of fresh (photo)catalytic
interfaces. In addition, photocorrosion would have continuously released Zn2+
ions, keeping their concentration at the illuminated ZnS surfaces high.
Besides reducing organic compounds and catalysing condensation reactions,
semiconductors can drive the photoreduction of dinitrogen to ammonium. This
reaction has been demonstrated with preparations of CdS [280] and TiO2 [281-
284]. The photoreducing capacity of ZnS is higher than that of CdS and TiO2
[154,190], and therefore one would expect that primordial ZnS systems were
capable of reducing dinitrogen to ammonium as well, thus complementing the
ammonium content of hydrothermal fluids. The interaction of formate, produced
upon CO2 reduction, with ammonium could yield formamide, which could serve
as a universal building block for the (photocatalyzed) synthesis of both nucle-
obases and amino acids [285].
Taken all together, these data support the suggestion that porous ZnS edifices,
when formed at the UV-irradiated surface of primordial Earth, could photore-
duce CO2 and other chemical compounds. On a geological scale, the productiv-
ity of this photosynthesis may have been immense.
First Settlers in the ZnS World

The suggested accumulation of complex but photostable π-systems in the UV-il-
luminated environments is in agreement with the presence of polycyclic aromatic
hydrocarbons in meteorites [194,286], as well as with the reported synthesis of
nucleotides from simpler precursors at the outer surfaces of Russian spacecraft
[287]. Generally, one would expect that complex organic compounds would
break into pieces in outer space. This is not the case; instead, the “pieces” join
together at surfaces to build complex aromatic π-systems that apparently are more
stable than simpler compounds against diverse types of cosmic radiation.
Senanayake and Idriss showed that TiO2 surfaces catalyzed a UV-powered
transition of formamide into nucleobases [196]. These results document the abil-
ity of seemingly “destructive” UV light to drive syntheses of increasingly complex
compounds at semiconducting surfaces.
Sutherland and co-workers have obtained activated pyrimidine ribonucle-
otides from cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde, and in-
organic phosphate in a reaction that bypassed free ribose. The synthesis yielded
activated ribonucleotide β-ribocytidine-2’,3’-cyclic phosphate as a major product
and several co-products [17]. Prolonged irradiation of this mixture by 254 nm
UV-light caused the destruction of various co-products and the partial conver-
sion of β-ribocytidine-2’,3’-cyclic phosphate into β-ribouridine-2’,3’-cyclic phos-
phate. The authors concluded that there must be some (photo)protective mecha-
nism functioning with the two natural nucleotides but not with other pyrimidine
nucleosides and nucleotides [17].
The reason for the particularly high photostability of nucleobases has re-
cently been clarified [164,165,288-293]. Two types of photochemical reaction
paths, which lead to an extremely fast transition from the excited state into the
ground state, have been identified, namely (i) the torsion of certain C-N bonds of
rings and (ii) the de-protonation of azine or amino groups [164,292,293]. Both
mechanisms require nitrogen atoms in the rings, which might explain the rather
counter-intuitive (for a CO2-dominated environment) selection of nitrogenous
compounds as constituents of RNA. More recently, it has been argued that the
high photostability of nucleobases is not affected by their alkylation; such alky-
lated, mostly methylated, derivates (known as minor nucleobases) are found in
the structures of tRNA and rRNA [294]. Together, major and minor nucleobases
could represent the initial pool of primeval photostable compounds from which
the major nucleobases were gradually selected by evolution [22].
To survive, the RNA-like oligomers should be resistant not only to solar UV
light but also to the (photo)chemical activity of ZnS itself. The reducing potential
of photoexcited ZnS is in the range of between -1.0 V and -2.0 V, depending on
the crystal structure [149,154,295]. This is low enough to reduce CO2 (see Fig.
1), but insufficient to reduce any of the nucleobases that all have reducing poten-
tials of less than -2.0 V [296,297]. On the other hand, as indicated by the energy
diagram of Fig. 1, the holes that are formed in photoexcited ZnS have an oxidiz-
ing potential of > 2 V [154,190] and can potentially oxidize almost any adsorbed
organic molecule, including nucleobases or nucleotides. Such an oxidation, how-
ever, is unlikely for two reasons. First, since ZnS is an n-type semiconductor,
the holes, unlike electrons, are not mobile [298]. Second, ZnS contains intrin-
sic electron donors, namely sulfur anions (S2-), which should “outrun” external
electron donors and reduce the immobile holes, yielding sulfur anion radicals,
S·-[298,299]. The formed S·-radicals could either dismutate according to eq. 2,
with the formation of disulfide anions [300], or they could be reduced by external
hole scavengers (see eqs. 3 and 4 and [278]), or they could interact with organic
compounds yielding their sulfo-derivates; in any case, however, they should not
be able to oxidize nucleobases or nucleotides that all have oxidizing potentials
above 1.2 V at neutral pH [301].
Photochemical damage from ZnS was also unlikely. Nucleobases absorb light
at 260–270 nm and emit it at 300–310 nm [159,302]. ZnS nanoparticles ab-
sorb light in a broad range up to approx. 350 nm and emit light at 420–470 nm
[144,303]. Therefore the radiation energy could be transferred from the adsorbed
nucleotides to a ZnS template (and thus contribute to the CO2 reduction), but
not in the reverse direction. Hence, the quanta directly absorbed by the ZnS tem-
plates would not damage the adsorbed RNA-like replicators.
Besides nucleobases, the primeval RNA-like polymers may have contained
ribose and phosphate entities. Ribose may have been abundant as one of the prod-
ucts of the autocatalytic “formose” reaction, which was discovered by Butlerov in
1861 [304] and which yields a mixture of pentoses and hexoses from formalde-
hyde. Although Butlerov’s reaction remains the only known autocatalytic reaction
that does not require specific catalysts, the importance of this reaction for prebio-
logical syntheses has been questioned since the yield of ribose in the product mix-
ture is usually low. Recent studies have shown, however, that the yield of ribose
can be selectively enhanced by the presence of phosphate in the reaction medium
[305], by UV illumination [16], and by conducting the reaction in the presence
of catalytic mineral templates [306]. More recently, it has been demonstrated
that the yields of pentoses increase to 60% and those of the ribose proper rise to
20% in the presence of a zinc-proline complex as a catalyst [15]. The Zn world
settings may have favoured autocatalytic ribose formation from photosynthesized
substrates by providing mineral templates, UV irradiation, and plenty of Zn2+
ions as catalysts.
It has been argued that biological stereoselectivity (homochirality), i.e. the

utilization of only particular optical isomers by living organisms, could have be-
gun from the selection of a particular D-isomer of ribose – since nucleobases
and phosphate groups are non-chiral [307]. Generally, homochirality cannot be
completely explained in the framework of the “primordial soup” concept [3-5],
because stereoisomers are chemically indistinguishable in a homogenous solution.
At a surface, however, the properties of two stereoisomers could differ [307], as
first pointed out by Goldschmidt, who has suggested that mineral surfaces were
involved as templates in abiogenesis [35,308]. The two other tentative mechanisms
of primordial stereoselectivity are (i) photoselection by polarized UV light (see
[309] and references therein), and (ii) enantiomeric autocatalysis (see [310,311]
for reviews). An example of this latter mechanism is the Soai reaction, where
heteroaromatic aldehydes react with organo-zinc compounds, yielding respective
alcohols, which in turn serve as asymmetric catalysts for their own formation. If
one of the substrate enantiomers is present even in small excess, the autocatalytic
reaction can yield the corresponding product with up to 95% enantiomeric excess
[312-314]. Although the mechanism of the Soai reaction remains unclear, Zn2+
ions might be important – zinc-proline complexes were also shown to mediate
stereoselective catalysis of aldole reactions in water [315]. At this point it is ap-
propriate to mention that the Zn world settings could support all these mecha-
nisms of stereoselectivity by providing (i) electrically charged surfaces with regular
patterns of positive and negative charges, (ii) Zn2+ ions that could build po-
tentially catalytic complexes with diverse organic compounds, and (iii) UV light
that would become polarized after passing through the ZnS crystals [316]. These
factors resemble strikingly the aforementioned features that increased the ribose
yield in the autocatalytic Butlerov’s reaction. Although the relative importance
of the above-named features for prebiological stereoselective and/or autocatalytic
syntheses of D-ribose and other sugars remains unclear, they can be experimen-
tally tested: the stereoselective (photo)catalysis of diverse organic reactions at the
surface of ZnS is an established approach in photochemistry [191,317-320].
Turning to the suggestion that the primordial waters were enriched not in phos-
phate but in the better soluble phosphite, it is worth noting that phosphate and/
or phosphite groups may have catalyzed prebiotic syntheses [17,32,305,321,322],
serve as bridges upon connecting nucleobases with metal sulfides [279,323], par-
ticipate in UV-driven photochemical reactions [324], prevent hydrolysis of the
first oligomers [325], and interact with diverse organic molecules, yielding their
phosphorylated derivates [252,326]. It is tempting to speculate that the oxidation
of phosphite ions, as hole scavengers, upon ZnS-mediated photosynthesis (see
above) may have been coupled with their involvement in polymerization reac-
tions as catalysts, yielding surface-confined oligomers connected by phosphate
groups [252,255,327].
The Zn world concept is consistent with a direct assembly of polynucleotides

at ZnS surfaces either from stacked nucleobases, ribose molecules, and phosphate/
phosphite linkers or, perhaps, even from simpler parts (as exemplified by Suther-
land and co-workers [17]). It is plausible that diverse (photo)synthetic pathways
may have been realized at the ZnS surfaces with the output being essentially de-
termined by photoselection of most stable compounds.
The light-induced energy transfer between adsorbed organic dyes, single nu-
cleotides, and polynucleotides on the one hand, and ZnS/CdS templates on the
other hand, has been intensively studied [242-244,247,328]. In one case at least,
it has been quantified that an organic dye fac tris(2-phenylpyridine) iridium,
when adsorbed on a ZnS surface, can serve as an antenna and increase the amount
of quanta captured by ZnS [329]. This result supports the possibility of a positive
feedback between the number of UV-light-absorbing zymes in a ZnS compart-
ment and the yield of photosynthetically produced metabolites.
DNA and RNA are capable of long-range energy transfer along stacked nucle-
obases, over tens of nucleotide pairs [330-333]. In the context of the primordial
Zn world, this property could have been useful: if a UV quantum hit a nucleotide
having no direct contact with an energy-absorbing template, then, owing to the
coupling between the stacked and paired nucleobases, the excess energy would
still have promptly sunk into the ZnS substratum.
The suggestion that RNA-like polymers could bind, via negatively charged
phosphate groups, to ZnS surfaces is supported by the ready adsorption of nucle-
ic acids on the ZnS/CdS nanoparticles [243,247,328,334-340] The assembly of
RNA-like polymers at the ZnS surface should be greatly facilitated by the comple-
mentary match between the patterns of electrically charged groups at the surfaces
of polynucleotides and ZnS, respectively. Such a matching was shown for the
CdS nanoparticles, which formed spontaneously from added cadmium salts in
the presence of polynucleotides as templates [341,342]. This match may have a
straightforward explanation: the distance between the positively charged Cd2+/
Zn2+ ions of 0.58/0.54 nm, respectively, in the nanoparticles [343,344] is simi-
lar to the distance of 0.58–0.59 nm between the phosphate groups in the RNA
backbone [345].
Wächtershäuser has suggested that a regular mesh of electric charges at the
surface may have made the primeval polymerization thermodynamically more
favourable by attracting reactants and arranging them appropriately relative to
each other [124]. De Duve and Miller have countered him by noting that if the
polymerization at the surface was thermodynamically more favourable than in the
bulk solution then the polymers could not detach without “paying” the respective
energy fee and would have remained confined to the surface [200]. As argued
above, such confinement would have prevented photodestruction of the polymer
molecules and favoured their interactions at the ZnS surfaces. It is worth noting
that a mechanism of thermodynamic confinement is actually exploited by nature
upon the synthesis of ATP from ADP and inorganic phosphate by membrane
ATP synthases. While the free energy of ATP synthesis in water is about +50 kJ/
mol, molecules of ATP build up spontaneously in the enzyme active site, the reac-
tion being facilitated by a positively charged arginine residue [346,347]. The mol-
ecules of ATP, however, remain tightly bound: they can leave the catalytic pocket
only after the free energy of membrane potential is used to open the pocket and to
reorient the arginine residue; ATP can then dissociate into the water phase [346].
The phosphate group in ATP is linked by a phosphoester bond that is similar to
those connecting the nucleotides in RNA and DNA. Hence, as compared to a
reaction in a bulk-water phase, the formation of phosphoester bonds can indeed
be favoured when the reactants are appropriately arranged and a positive charge
is present nearby.
Orgel and co-workers have studied the polymerization of guanosine 5’-phos-
phorimidazolide on a polycytidylic acid template in the presence of a variety of
metal salts [198,348]. They found that “none of the metal ions investigated be-
haved like Zn2+ in favoring the formation of the naturally occurring 3’-5’ link-
ages” (quoted from [198]). A specific role of Zn2+ ions in shaping the proper
3’-5’ linkages follows also from the recent work of Hadley and co-workers. They
selected deoxyribozymes that could ligate RNA and found that the native 3’-5’
linkages were built only by those deoxyribozymes that were dependent on zinc
[349]. In addition, mostly 3’-5’ bonding has been observed upon the radiation-
driven polymerization of nucleotides at the surface of volcanic rocks [350]. Hence,
both Zn2+ ions and mineral templates seem to favour the formation of proper
3’-5’ bonds.
The emergence of longer RNA strings could have proceeded not only via po-
lymerization but also through spontaneous rearrangements of RNA sequences
that may progress in the absence of any enzymes or ribozymes [351-353]; such
rearrangements may have dramatically accelerated evolution [57,354].
In sum, the (photo)catalytic properties of Zn2+ ions and Zn-containing sub-
stances could have shaped the first life forms. While the photochemistry of ZnS
crystals could have governed the nature of photosynthesized compounds and that
of their photo-derivates, the catalytic properties of Zn2+ ions may have deter-
mined the particular traits of the first (bio)molecules, such as the choice of 3’-5’
linkages for RNA polymers.
The First Replicators and the Emergence of Proteins and

Enzymes
Earlier, while hypothesizing on the selective advantage of paired RNA strands
over those unpaired in primordial UV-illuminated settings [112], we built on
empirical evidence of the higher UV stability of double-stranded RNA samples as
compared to single-stranded ones [204-206]. Recently, the physical background
of this higher photostability has been clarified. For the nucleotides that form a
Watson-Crick pair, the lifetime of the excited state has been estimated to be as
low as a few femtoseconds [355,356], i.e. ca. one hundred times shorter than that
of single bases [159-163]. This extremely short lifetime has been attributed to
excited-state deactivation via electron-driven proton shuttling between the bases
[357-359]. It is noteworthy that other possible (not Watson-Crick) conformers of
paired nucleobases have not shown these unique photochemical properties [355].
It has been suggested “that the biologically relevant Watson-Crick structures of
GC and AT are distinguished by uniquely efficient excited-state deactivation
mechanisms which maximize their photostability” (quoted from [164]).
It is necessary to emphasize that neither the high photostability of single nu-
cleobases [159-165,288-293], nor the even higher stability of their Watson-Crick
pairs [164,355,356], nor the aptitude for long-range energy transfer along stacked
nucleotides [330,333,360] have anything to do with the current functioning of
RNA and DNA in the transfer of genetic information. All these traits, however,
support the suggested involvement of UV light as a selecting factor during the
initial stages of evolution [105,112,133,159,164]. This selection pressure would
have favoured the relative enrichment of photostable aggregates built of paired
RNA-like strands with a potential for self-replication.
The idea of primeval RNA-based self-replicating aggregates got additional
support from the work of Lincoln and Joyce who presented a system of two ri-
bozymes that catalyzed synthesis of each other from a total of four oligonucle-
otide substrates. These cross-replicating RNA enzymes underwent self-sustaining
exponential amplification in the absence of proteins or other biological materials
[72].
The data on specific affinity of various proteins to the ZnS/CdS nanoparticles
[243,246,361-364] suggest that ZnS surfaces could serve as templates for the
synthesis of the first polypeptides or as baseplates for the first protein synthases.
A primeval RNA machine capable of making peptide bonds has been recently
unveiled by Bokov and Steinberg based on a detailed analysis of the structure of
the modern ribosome [73]. As the first protein synthases most likely began by
generating random polypeptides [73], it might be useful to consider – in a search
for tentative sequence-independent functions of the first proteins – the traits that
are common for all polypeptides. To perform sequence-independent functions,
the first polypeptides should have been able to interact with polynucleotides via
their backbone atoms. Carter and Kraut have suggested that a protein chain can
fit into the minor groove of an RNA helix, with hydrogen bonds being formed
between the ribose 2’-hydroxyls and carbonyl oxygen atoms of peptide bonds, in
an interaction that is RNA-specific and is not possible in the case of DNA [365].
Indeed, hydrogen bonding between the protein backbone oxygen atoms and the
ribose 2’-hydroxyls has been found in many RNA-protein complexes [366]. This
ability of proteins to block the 2’-hydroxyls of ribose entities may have increased
the life span of primordial RNA polymers. The aforementioned ability of Zn2+
ions to catalyze the formation of proper 3’-5’ linkages [198,349] implies the abil-
ity of Zn2+ ions also to catalyze cleavage of these linkages. Butzow and Eichhorn
have noted that the Zn2+-catalyzed cleavage of RNA starts from the binding of
a Zn2+ ion between the 2’-hydroxyl group of ribose and the phosphate group
[367]. It is tempting to speculate that the first polypeptides could protect the
RNA molecules from metal-catalyzed cleavage by preventing the binding of Zn2+
ions to the 2’-OH groups of ribose.
The (photo)stability of polynucleotides and proteins, as argued by Sobolewski
and Domcke [164], is ensured by reversible proton relocation within picosec-
onds between the Watson-Crick-paired nucleotides and along the protein hy-
drogen bonds, respectively. In other words, the reshuffling of protons along hy-
drogen bonds can promptly split a large “captured” energy quantum into many
small, non-hazardous heat quanta, both in nucleic acid polymers and in proteins
[164]. In a UV-irradiated environment, this feature should favour the selection
of structures with many intrinsic hydrogen bonds, such as paired RNA or DNA
strands, protein α-helices and β-sheets, as well as hydrogen-bonded RNA-protein
aggregates. Turning to the previously discussed relation between the stability of
polymers and their ability to perform work, the shuttling of protons along hy-
drogen bonds might be considered as a kind of work that could be carried out
at picoseconds, i.e. much faster than any bond dissociation could take place. It
is noteworthy that the acid-base catalysis, seemingly prevalent in ribozymes and
enzymes [368-371], consists of proton relocation(s) between the donor and ac-
ceptor groups [372-374]. Accordingly, the selection of hydrogen-bonded systems
as more (photo)stable ones could have paved the way to the first catalytic centres,
first in ribozymes and then in proteins.
Several authors, based on quite different premises, have argued that the first
genetically coded amino acids were the neutral and acidic, starting from glycine,
alanine, valine, and aspartate, with the positively charged amino acids being added
later [46,47,53,375,376]. In the absence of positively charged amino acids, metal

ions and, in particular, Zn2+ ions, could have been recruited by the first enzymes
as catalytic Lewis acids (Manfred Eigen, personal communication).
In summary, the illuminated ZnS settings may have contributed to the emer-
gence of first replicators and enzymes by favouring the formation of photostable,
hydrogen-bonded structures, by serving as templates upon syntheses, and by pro-
viding Zn2+ ions as potent catalytic cofactors.
Colonization Waves
Chetverin and co-workers have introduced and studied RNA colonies that grew
and propagated on gels or other solid media provided that RNA replicases and
ribonucleoside triphosphates were present [351,377,378]. It has been explicitly
noted that such experimental systems might, in fact, model the amplification
and propagation of the first replicators in primordial environmental settings
[57,351,354]. Further indications of primeval RNA life might be the participation
of tRNA molecules as catalysts in several metabolic reactions (see [23] and refer-
ences therein) and interactions of RNA molecules with metabolites [379,380], in
particular in the case of riboswitches [381,382].
As argued by Koonin and co-workers, viral hallmark genes shared by many
groups of RNA and DNA viruses – but missing in cellular life forms – might be
relics from the pre-cellular RNA/protein world [61]. Moreover, the specific affin-
ity of many metabolic enzymes to RNA [383,384] could also stem from the life
forms that were built of RNA and proteins.
Last but not least, porous, inhabited ZnS settings still persist around deep-sea
hydrothermal vents; their dwellers, mostly archaea, have been characterized on
several occasions [179,181].
At the end of this chapter, it is fair to note that a large part of the cited evi-
dence comes from nanotechnology research in which ZnS/CdS nanoparticles are
paradigmatic objects of study. Still, while sifting through the literature on the
interaction of nucleobases, polynucleotides, and proteins with ZnS/CdS nano-
particles (see e.g. [242-244,246,247,328,334-342,361-364,385]), it is difficult
to avoid the impression that the “intrinsic affinity of (poly)nucleotides for semi-
conductor surfaces” (quoted from ref. [334]) is particularly specific. In the frame-
work of the Zn world scenario, it is tempting to speculate that, while interact-
ing keenly with the ZnS/CdS surfaces, the biological polymers might recall their
evolutionary past.
Evolutionary Continuity in the Zn World

A scenario for the evolution of a complex system must consist of plausible ele-
mentary steps, each conferring a distinct advantage (Darwinian continuity princi-
ple; see also ref. [27,67,218]). Furthermore, these steps also have to be physically
plausible, which implies, in addition to the correspondence with physical laws,
the continuity of underlying mechanisms and driving forces. Below, the interplay
between the Darwinian and physical continuities in the Zn world is considered
in more detail.
Multifarious Energetics of the Zn World

Admittedly, the least physically plausible step in the available origin of life sce-
narios is the abiogenic emergence of complex polymers capable of replication
and catalysis. As discussed above, at least one type of polymer should have been
continuously emerging under primeval settings to enable a selection of first cata-
lytic entities that could gradually develop an ability to synthesize other types of
polymers. Based on the aforementioned arguments, these primary polymers could
be related to modern RNA. The synthesis of RNA and DNA molecules in mod-
ern organisms is driven by the hydrolysis of ATP molecules and carried out by
sophisticated enzyme systems. Turning to the primordial Earth, one has a typical
“chicken and egg” paradox: on the one hand, high-yield polymerization would
seemingly have required specific machinery for coupling with exergonic reactions,
yet, on the other hand, this machinery was likely to have been absent before the
first catalytic polymers have emerged. One of the virtues of the Zn world scenario
is the possibility to funnel UV energy into polymerization reactions. In addi-
tion, the thermodynamically very favourable oxidation of phosphite to phosphate
could potentially provide free energy. Furthermore, the polymerization at ZnS
surfaces could be more favourable than in the bulk-water phase (see above). Nev-
ertheless, it is unclear whether these factors alone could have been sufficient for
maintaining a notable steady-state population of RNA-like polymers – needed
as starting material for further evolutionary transformations. The results of our
earlier Monte-Carlo simulations of primordial photochemistry [112], suggest one
more, rather paradoxical, way to channel external energy into the synthesis of
increasingly complex compounds.
The aim of the simulations was to quantify the significance of the UV protec-
tion mechanism for the evolution of primordial RNA-like polymers by computer
modelling of the polymerization of sugar-phosphate monomers in the presence
of nucleobases.
1) “Dark” simulations (with “virtual” UV light switched off and a polymer-

ization constant of > 1) yielded polymers that, as expected, did not carry
nucleobases, since the equilibrium constant of their attachment to the
sugar-phosphate units was taken, in accordance with the actual situation,
as <<1 (see [112] for further details). Despite a high polymerization con-
stant, the elongation of polymers was not unconstrained. The explanation
as suggested by D. Cherepanov (personal communication) is the follow-
ing: if a linear polymer contains n monomers, the binding of new mono-
mers can proceed only at the two terminal positions, while dissociation
can implicate any bond, i.e. it can proceed at (n-1) positions. Hence, the
probability of dissociation should increase with the value of n and prevent
the formation of excessively long polymers. This straightforward consid-
eration, as well as the simulation results, discount the common belief that
the longer polymers could have built up solely by means of their postu-
lated higher stability against hydrolysis as compared to shorter polymers
(see e.g. [386]).
2) After turning the virtual UV light on and in the absence of UV protec-
tion, with UV quanta being able to cleave the sugar-phosphate bonds,
the polymerization yield dropped severely, and the extent of nucleobase
incorporation in the polymers remained close to zero.
3) With the UV protection “switched on” (so that the binding of a nucle-
obase to a sugar-phosphate unit decreased the probability of UV breakage
by a factor of 30), the length of formed polymers increased dramatically.
Even more importantly, under these conditions the fraction of nucleobase-
carrying sugar-phosphates increased significantly (up to ~0.5; see [112]).
4) We also simulated the effect of the partial funnelling of UV energy into
the condensation reactions by assuming that with a probability as small
as 9 × 10-8 a photogenerated radical could bind a nucleobase in a proper,
productive way. The resulting boost for the formation of oligonucleotides
was remarkable concerning the length of the formed polymer chains and
the extent of nucleobase incorporation into the oligomers, so that the long
polymers were built predominantly from nucleobase-carrying sugar-phos-
phate units (i.e. nucleotides; see [112]).
Result no. 4 is physically trivial because it follows from the enabled channel-
ling of the virtual solar energy into the synthetic reactions. However, the increase
in the relative fraction of longer, nucleobase-carrying polymers solely in response
to switching on the UV protection (result no. 3) is not trivial at all, because in this
case the utilization of radiation energy for synthesis was not “permitted”. Here,
the increase in the fraction of complex polymers was due to the breakage of the
less photostable molecules by the virtual UV light. Since the number of molecules
in the Monte-Carlo simulation was limited, the relative fraction of more photo-
stable and, accordingly, more complex polymers increased. Therefore – and this is
the key point – the enrichment in more complex RNA-like polymers was driven
by the photo-dissociation of their less stable counterparts, in the absence of any
direct coupling between the energy flow and the synthetic reactions.
From the experimental viewpoint, the results of this modelling might be re-
lated to the aforementioned finding of Sutherland and co-workers that prolonged
UV illumination of the reaction medium containing a set of pyrimidine nucle-
otides and nucleosides led to the enrichment of photostable natural nucleotides
on account of non-natural compounds [17]. From the theoretical viewpoint, the
described mechanism of “indirect” energy utilization might be related to the so-
called Landauer principle. Rolf Landauer, while working for IBM, demonstrated
that, contrary to common belief, information per se could be created in a revers-
ible way, i.e. “for free”. Further analysis has shown, however, that energy would
then be required to erase all the intermediate steps of such a reversible computa-
tion from the computer memory [387-390]. Accordingly, the creation of infor-
mation requires energy in any case, but the energy can be utilized in two different
ways: (i) for creating the information itself and/or (ii) for erasing the “garbage”
from the system. It is noteworthy that the need for “erasing energy” arose because
Landauer had considered a physically realistic computer with a limited number
of memory units. At least on this point, Landauer’s formalism corresponds to our
Monte-Carlo simulation that also invoked a limited number of building blocks
[112].
Several authors have argued recently that the physical model of Landauer
might be related to biology, in particular to creating and maintaining increasingly
complex life systems [92,391-393]. As noted by Danchin, “creation of informa-
tion requires many steps, it starts from a given complex of dynamic interacting
entities, that progressively transforms into variants, among which some have a
higher information than that of the original complex” [92]. It would seem that
this quotation is equally applicable to computation processes and to the evolution
of first RNA-like polymers on primordial Earth. Then, however, the emergence
of increasingly complex RNA-like polymers could be driven by the energy-con-
suming, selective erasing of less “perfect” variants. Danchin writes that this type
of selection process has to “actively discriminate between entities that are in some
degree functional and those that cannot function. This is because the process
needs to avoid destroying the elements that carry increased information, and this
is where energy comes in. Energy has to be consumed to make innovations stand
out, in a discriminant process: energy is used to prevent degradation of functional
entities, permitting destruction of the non-functional ones” (quoted from [92]).
It is noteworthy that Danchin has invoked Landauer’s formalism to describe the

aging phenomena. Still, this approach seems to be applicable to the primordial
RNA-like polymers in the Zn world, provided that we consider the more photo-
stable constructs as “functional” ones and the solar UV radiation as the selecting
and erasing energy input. In fact, since the more complex, longer polymers have
more opportunities to fold or aggregate with the formation of UV-stable double
strands, the increase in photostability should be coupled with the increase in com-
plexity and, accordingly, in the information content (see also below). Incidentally,
the interplay between supporting and erasing energy fluxes, which is apparent in
the case of photostable RNA-like molecules, might represent the physical essence
of biological evolution (see [92,394,395] for related discussions).
In any case, the competition between the RNA-like oligomers should have
been tough in the primeval Zn world. The ZnS compartments (pores) could ac-
commodate only a limited number of polymers, so that the enrichment of more
photostable polymers – driven by the photo-dissociation of the less fit ones –
must have been pronounced. In addition, the survival of a molecule depended not
only on possession of apt UV quenchers but also on binding to a mineral template
capable of absorbing excess radiation energy, so that primordial polymers would
have competed for free surface patches. In this competition, the RNA-like poly-
mers that, as argued above, could match the ZnS surfaces electrostatically would
have gained an advantage.
It is possible to conclude that in the Zn world the energy of solar UV radiation
may have been utilized in three different, although complementary, ways, namely:
(i) for a high-yield, ZnS-mediated photosynthesis of diverse organic compounds,
(ii) for fortuitous photopolymerization events, and (iii) for relative enrichment of
the photostable, surface-adsorbed RNA-like polymers via the systematic photo-
dissociation of more labile molecules.
Reproduction and Replication in the Zn World

A few authors have noted that the origin of life could be a two-step process,
with the emergence of the first replicators being facilitated by the pre-existence of
out-of-equilibrium settings favouring their formation (see e.g. [27,396,397] and
references therein). Dyson has hypothesized that the emergence of the first life
forms capable of error-free replication may have been preceded by simpler repro-
ducing systems where interacting chemical cycles, supported by external energy
sources, could reach homeostasis and persist over time. He has even speculated
that such reproducing systems could have evolved and attained complexity com-
parable to that of modern cells [398]. The Zn world settings can be considered
out-of-equilibrium and reproducing, with their persistence being guaranteed by
the hydrothermal activity of Earth and by solar energy. The precipitation of ZnS
around hot springs would have led to a continuous reproduction of new, empty
compartments [61,176,219] where the selection of replicators could proceed
on a geological timescale. In such settings, photostable compounds could have
continuously emerged and undergone photoselection for millions of years, until
their particular mutual interaction yielded an aggregate capable of robust self-
replication. The emergence of life can therefore be considered not as an accident,
but as a natural outcome of the interplay between solar radiation and particular
geothermal processes on primordial Earth.
The role of reproducing, illuminated ZnS settings may have been in enabling
the (photo)selection of the first replicating entities. Hence, this solar-powered
selection may have been a link between abiogenic reproducing systems and the
first replicators.
The Origin of Meaningful Complexity in the Zn World

Since the organisms, and not the polynucleotide molecules, are the subjects of
evolution, some authors have noted that nucleotide strings (e.g. the genes that
code particular proteins) are meaningless unless the machinery that translates
the DNA/RNA-coded information into the final products is explicitly taken
into account, see [399-401]. The Zn world scenario suggests how the biologi-
cal meaning could emerge gradually, being driven by (photo)selection, with the
more photostable systems gaining selective advantage upon each step. Indeed, if
we could trace, in a UV-irradiated environment, the fate of two RNA-like poly-
mers of the same length and the same information content (see [402,403] for
surveys of relevant definitions), we may find that the polymer that could fold
into a structure with more double-stranded segments and/or to bind tightly to an
energy-absorbing template could endure more hits by UV quanta. Therefore each
polymer could be characterized by its (i) intrinsic complexity related to the prob-
ability of its emergence [402,403] and (ii) extrinsic complexity, the value of which
would reflect its survival chances in given settings and could change depending
on conditions. The more stable constructs, with higher complexity, would have
had longer lifetimes and be steadily present in relatively larger numbers. Hence,
they would have had more chances to interact with other stable constructs to form
even more elaborate aggregates. At the moment when one such aggregate begins
to replicate itself, the complexity of its constituents, thus far related to their sur-
vival chances in a UV-irradiated environment, becomes biologically meaningful.
At the very same moment, the nucleobases – which were hitherto acting solely as
efficient UV-protecting units – become the letters of the genetic alphabet. In this
framework, the “meaning”, in the biological sense, is intrinsically coupled with
the survival chances under given circumstances and thus can precede the emer-
gence of the translation machinery (that could have gradually developed later; see
e.g. [46,47,58,67,73] for tentative scenarios).
Since polynucleotides do not last long in nature, replication can be considered
as a way to preserve (accumulate) information before an information carrier – a
polynucleotide strand – eventually breaks down because of thermal fluctuation or
(photo)cleavage. It is likely that the first replicating systems invoked linear poly-
mers because the replication of branched polymers – as a logical alternative – may
have been mechanistically impracticable.
In summary, we have an example of a Darwinian function co-option [404]:
the “irreducible complexity” of the first replicators can still be reduced by suggest-
ing that their simpler “ancestors” underwent selection for a different, less struc-
turally demanding aptitude (namely for their ability to survive in a UV-irradiated
environment).
Physical Continuity of Life

Living organisms are routinely considered as systems that sustain themselves by con-
verting “few” high-quality energy quanta into “many” low-quality, thermal quan-
ta, this process being accompanied by entropy production, see [87,90,209,405].
These high-quality (or high-energy) quanta are those that contain portions of
energy large enough to drive chemical reactions, such as the quanta of light in the
UV and visible ranges or the energy portions that are released upon redox reac-
tions. In such a framework, biological evolution could be understood as a way to
optimize the utilization of high-energy quanta [90,209].
The evolution of energy conversion at the earliest evolutionary stages, e.g.
in the RNA world, as well as the driving force(s) beyond this evolution have re-
mained, however, undefined. In modern organisms, the conversion and storage
both of the energy of light and of chemical energy are carried out by membrane
enzymes that catalyze sophisticated chains of redox reactions (see e.g. [176,406-
408] for reviews). It is noteworthy that (known) natural ribozymes cannot store
either the energy of light or the redox energy [68]. Not surprisingly, the tradi-
tional concept of the RNA world has circumvented the question of exploiting the
external energy fluxes.
Before addressing this question, it is useful to realize that the ability of high-
energy quanta to drive chemical reactions does not require these reactions to
be useful. For a living organism, the outcome from capturing a UV quantum
may have been hazardous rather than beneficial. In the Zn world, where surface-
confined photosynthesized molecules could not escape solar UV light, the first
RNA-like polymers may have initially been selected not for their ability to exploit
the high-energy quanta but for the ability to discard them promptly or, in other
words, to “process” them without undergoing photodamage. The ability to dis-
card the energy quanta is less structurally demanding than the ability to exploit
them, so, as argued above, this ability may already have been inherent in simple
organic molecules. It is tempting to suggest that the common energetic denomi-
nator of living organisms – throughout the whole evolutionary time span – may
have been, not the ability to exploit high-energy quanta, but the ability to “deal”
with them – in a general sense.
This suggestion makes it possible to follow a continuous evolutionary thread
from the origin of life event to the modern struggle of mankind for energy. At
the beginning of this thread, the first RNA-like polymers could have emerged as
systems capable of discarding potentially hazardous high-energy quanta without
undergoing damage. The energy requirements of the first replicators could have
been covered by the breakdown of “nutrients” produced in photosynthetic but
abiogenic reactions. The first heterotrophic organisms, while retaining the ability
to discard excess energy, could have gradually developed abilities to control the
high-energy quanta and to use them. It is plausible that solar energy, besides serv-
ing as a selective factor, may have occasionally promoted chemical reactions (e.g.
as happens in natural photolyases [409,410] or in an artificial deoxyribozyme
with photolyase activity [411]). The funnelling of high-energy quanta into use-
ful reactions may have been profitable per se and, in addition, could have pre-
vented undesirable overheating and denaturation. However, only the emergence
of biogenic dielectrics, namely proteins and phospholipid membranes, enabled
the storage of the energy of light in the form of charge-separated states, physically
analogous to those involved in ZnS-mediated photosynthesis (see Fig. 1 and the
accompanying article [226]). This development may have paved the way to the
first phototrophic organisms in which membrane-embedded, energy-converting
systems could have developed, supposedly, from the UV-protecting membrane
proteins of the first cells [226,412]. It is noteworthy that modern chlorophyll-
carying proteins, besides being involved in the storing and utilization of solar
energy, retain the ability to protect cell DNA from the residual UV component
of solar radiation [413], thus resembling the nucleobases that, in addition to serv-
ing as the letters of genetic code, continue to protect DNA and RNA backbones
from UV damage [159]. In this framework, the gradually attained ability to ex-
ploit high-energy quanta of light can be considered as an evolutionary younger,
as compared to their deactivation, and more sophisticated way of dealing with
them. As the accessible energy assets ran out, living organisms had to “acquire
new powers” (quotation from [1]), i.e. increase their complexity, if they wanted to
access and exploit new energy sources (see [21,90,209] for detailed considerations
of this point).
In summary, the emergence of life on Earth may be traced to the successful

attempt of particular (hetero)cyclic molecules to overcome, in a joint effort, the
hazards of solar UV rays that they could not escape.
Relationship to Other Hypotheses on the Origin of Life

The suggested scenario invokes elements from different, even seemingly contra-
dicting, hypotheses on the origin of life. This should not be surprising, since each
of these hypotheses builds upon empirical data and/or certain theoretical bases.
Accordingly, these empirical observations and theoretical considerations have to
be accounted for by any scenario that claims to serve as a platform for further
discussions in the field.
Oparin’s hypothesis of a heterotrophic origin of life implied that the initially
established metabolism could have resembled fermentation [3,4]. The rationale
behind this supposition (see [26,28,29,34] for further development of this view)
was straightforward: fermentation is a simple mode of metabolism that is in-
herent in the most primitive anaerobic organisms. We now know that catalytic
chemistry beyond the breakage or formation of covalent bonds is indeed the least
structurally demanding; in most cases only acid-base catalysis is involved, often
enhanced by metal atoms [368-371]. Not surprisingly, reactions of this type can
be catalyzed even by ribozymes, unlike many other chemical transformations that
can be performed only by proteins. The Zn world scenario builds on this rationale
and suggests that primordial metabolism, as catalyzed by the first ribozymes and
enzymes, constituted a set of chemically simple transformations where reactions
of bond breakage were coupled with reactions of bond formation, perhaps being
mediated by group transfer events. Oparin, however, did not treat primordial
energetics explicitly [3,4]. The formation of the first polymers was suggested to
proceed spontaneously, without coupling to energy flow. Without such coupling,
however, an unsupported formation of long polymers would contradict the laws
of thermodynamics, as routinely noted by critics of Oparin’s idea (see e.g. [83]).
In contrast, the Zn world scenario invokes solar UV light as an energy source both
for prebiological syntheses and for (photo)selection.
Haldane, in addition to a fermentation-like metabolism, which he postulated
independently of Oparin, suggested that “the first living ... things were probably
large molecules synthesized under the influence of Sun radiation” (quoted from
[5]). Hence, on this point Oparin’s and Haldane’s hypotheses differ. In addition,
Haldane argued that the complexity of the first organisms should be compatible
to that of bacteriophages, thus anticipating some aforementioned modern views
related to the RNA world concept [61,62,219]. On all these points, there is an
agreement between the Zn world concept and Haldane’s hypothesis. Nonetheless,
Haldane suggested that life emerged in the homogenous phase (it was he who
mentioned the “hot, dilute soup”). In contrast, the Zn world concept considers
primeval life as a surface-confined phenomenon.
The Zn world concept is in accord with the RNA world (RNA life) hypothesis
[37-73]; the RNA-based life forms may have inhabited the photosynthesizing,
porous ZnS edifices that could help to (photo)select first RNA organisms, provide
a shelter and nourish them. RNA organisms most likely inhabited the Zn world
only in the beginning, being followed by RNA/protein life forms. Concerning
RNA research, the involvement of ZnS surfaces suggested here implies that, upon
modelling or simulating primeval events, electrically charged ZnS surfaces may
be explicitly invoked as supporting (photo)catalytic templates (see also [414]).
With their assistance, replication could have been carried out by relatively simple
aggregates of RNA-like polymers. In addition, the first systems of protein synthe-
sis, owing to supporting ZnS templates, may have been fundamentally simpler
than their modern counterparts (see also [73]). The gradual decoupling from the
ZnS surfaces and the emergence of enclosed organisms would have required more
complex devices, such as primeval ribosomes, which, however, had time to evolve
gradually from more simple predecessors – by recruiting new parts to functionally
replace the ZnS baseplates.
The Zn world scenario incorporates elements from some “metabolism first”
concepts, in particular from the “Pyrite world/Pioneer organism” concept of
Wächtershäuser, who put forward a detailed concept of a two-dimensional pri-
mordial life on iron disulfide (FeS2, pyrite) surfaces at the sea floor [124-130].
The Zn world scenario exploits the suggestion of Wächtershäuser that the mineral
surfaces may have promoted the abiogenic syntheses of the first polymeric mol-
ecules. In addition, the Zn world model builds upon the idea of Russell and co-
workers on porous chimneys of the deep-sea hydrothermal vents as incubators for
the first life forms [115-123]. In contrast with these two concepts, the Zn world
scenario considers zinc sulphide – instead of iron sulfides, which can generate po-
tentially harmful hydroxyl radicals [223,224] – as the “mineral of life”, and locates
the first life settings at the illuminated surface of Earth. Moreover, the suggested
metabolism in the Zn world, which combines abiogenic photosynthesis with the
primitive heterotrophy of the first replicators, is fundamentally different from
the (bio)chemical mechanisms that were postulated for the two “iron-sulfide”
worlds and based upon iron-catalyzed redox reactions (see the accompanying ar-
ticle [226] for more details on this point).
Kuhn and Waser have noted that the pores of primordial rocks could have
served as compartments upon the evolution of the first RNA molecules [45]. A re-
lated attempt to merge the FeS compartments with the RNA life has recently been
performed by Martin and co-workers [117,219], who have suggested that “within
a hydrothermally formed system of contiguous iron-sulfide (FeS) compartments,

populations of virus-like RNA molecules, which eventually encoded one or a few
proteins each, became the agents of both variation and selection” (quoted from
[219]). Experimental evidence of polynucleotide accumulation in simulated pore
systems has been provided by Braun and co-workers [415]. The idea of porous
natural settings as incubators for the first RNA-like life forms is used in the Zn
world scenario. It, however, suggests that the function of “honeycombed” ZnS
settings was not limited to compartmentalization but also included photosynthe-
sis of metabolites, (photo)selection of most fit polymers, and (photo)catalysis of
diverse reactions. In addition, the Zn concept implies that colonization of these
settings by first life forms proceeded from the illuminated, photosynthesizing top
to the bottom, in contrast to the scheme of Martin and co-workers in which life
propagated from the bottom, “secure” part of the deep-sea hydrothermal FeS vent
to the surface.
A few authors [19,416,417] have stressed the potential importance of high
radioactivity on primordial Earth for the origin of life, albeit without suggesting
how the energy of ionizing radiation could be used for biochemically relevant
syntheses. The Zn world scenario incorporates such a mechanism: ZnS crystals
can trap and transiently store, in a form of charge-separated states, diverse kinds
of radiation energy, namely X-rays, electrons (as in displays), α-particles (ZnS
was introduced as the first inorganic scintillator by Sir William Crookes in 1903),
and so on. The excited electron(s) could then drive the CO2 reduction at the
surface of ZnS crystals. Hence, ZnS crystals show a unique ability to convert
diverse kinds of energy into the chemical energy of organic compounds. Taking
into account the high radioactivity level on primordial Earth, the ZnS edifices
would have worked as natural converters of radioactive energy into reduced car-
bon and nitrogen-containing metabolites. The high-energy radiation could have
penetrated deeper into the ZnS edifices than the solar UV light and, in addition,
could have supported the ZnS photoreactors during the night hours.
Nonetheless, it is necessary to emphasize that the major source of energy for
primordial life was solar light, since its energy, according to available estimates,
was many orders of magnitude larger than that of ionizing radiation (see e.g.
[19,20,34,109]). In addition, the unique photostability of polynucleotides in-
dicates that they were shaped by evolution under the selective pressure of solar
light.
Conclusion
The suggested Zn world scenario (i) identifies the geological conditions under
which “primeval caves” made of photosynthesizing nanoparticles could emerge
and persist on primordial Earth, (ii) comprises a mechanism by which these par-
ticles could use “warm sun-beams” for the production of diverse organic com-
pounds, (iii) attributes the selection of primordial RNA-like polymers to their
particular photostability, and (iv) indicates the driving forces and selective factors
that might have promoted the transition from the first simple polymers to more
complex living organisms.
The Zn world scenario shows how the “metabolism first” and “replication
first” concepts can be reconciled and combined. The scenario comprises a mecha-
nism of the continuous abiogenic photosynthesis of metabolites and their further
conversion by ZnS-confined replicating entities.
The suggested concept invokes (photo)selection as a driving force – in the
straightforward physical sense – in the emergence of increasingly complex bio-
polymers.
In addition, the Zn world scenario is detailed enough to enable falsification
tests, as exemplified in the accompanying article [226].
Last but not least, the idea that life may have emerged within photosynthe-
sizing, luminescent crystals – which gathered the light of the harsh Hadean Sun
during the day and glittered through the nights – is aesthetically appealing.
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On the Origin of Life in the
Zinc World: 2. Validation
of the Hypothesis on the
Photosynthesizing Zinc
Sulfide Edifices as Cradles of
Life on Earth
A. Y. Mulkidjanian and M. Y. Galperin
Abstract
Background
The accompanying article (A.Y. Mulkidjanian, Biology Direct 4:26) puts
forward a detailed hypothesis on the role of zinc sulfide (ZnS) in the ori-
gin of life on Earth. The hypothesis suggests that life emerged within com-
partmentalized, photosynthesizing ZnS formations of hydrothermal origin
(the Zn world), assembled in sub-aerial settings on the surface of the prime-

val Earth.
Results
If life started within photosynthesizing ZnS compartments, it should have
been able to evolve under the conditions of elevated levels of Zn2+ ions, by-
products of the ZnS-mediated photosynthesis. Therefore, the Zn world hy-
pothesis leads to a set of testable predictions regarding the specific roles of Zn2+
ions in modern organisms, particularly in RNA and protein structures relat-
ed to the procession of RNA and the “evolutionarily old” cellular functions.
We checked these predictions using publicly available data and obtained ev-
idence suggesting that the development of the primeval life forms up to the
stage of the Last Universal Common Ancestor proceeded in zinc-rich settings.
Testing of the hypothesis has revealed the possible supportive role of manga-
nese sulfide in the primeval photosynthesis. In addition, we demonstrate the
explanatory power of the Zn world concept by elucidating several points that
so far remained without acceptable rationalization. In particular, this con-
cept implies a new scenario for the separation of Bacteria and Archaea and
the origin of Eukarya.
Conclusion
The ability of the Zn world hypothesis to generate non-trivial veritable pre-
dictions and explain previously obscure items gives credence to its key postu-
late that the development of the first life forms started within zinc-rich for-
mations of hydrothermal origin and was driven by solar UV irradiation. This
concept implies that the geochemical conditions conducive to the origin of life
may have persisted only as long as the atmospheric CO2 pressure remained
above ca. 10 bar. This work envisions the first Earth biotopes as photosynthe-
sizing and habitable areas of porous ZnS and MnS precipitates around pri-
meval hot springs. Further work will be needed to provide details on the life
within these communities and to elucidate the primordial (bio)chemical re-
actions.
Background
Energetic Aspects of the Origin of Life
The problem of origin of life on Earth (abiogenesis) remains one of the central
and most intractable problems of modern biology. The current hypotheses cluster
either around the “replication first” paradigm or the “metabolism first” concept,
see [1-10] for consideration of the controversy between the two concepts. The
“replication first” paradigm implies that formation of the first replicating entities
preceded the origin of metabolism. This concept has grown from the so-called
heterotrophic theory of abiogenesis that can be traced to Oparin, who had sug-
gested that formation of complex proteinaceous complexes could proceed spon-
taneously under the conditions of reducing primordial atmosphere [11,12]. The
Oparin’s proposal, which was the first detailed scenario of abiogenesis, found an
experimental confirmation. It has been shown later that simple building blocks,
such as amino acids and carbohydrates, indeed, could build up from inorganic
compounds under the conditions imitating the reduced primeval atmosphere,
provided that external energy was delivered in the form of electric discharges or
UV light [13-16]. The modern successors of Oparin’s hypothesis are various RNA
World scenarios, where the first RNA-like molecules are seen as capable both of
self-reproduction and simple metabolism and thus preceding both proteins and
DNA [17-33]. The “replication first” concept has been further supported by iso-
lation and characterization of RNA enzymes (ribozymes) with different catalytic
activities (see [27,29,31,34,35] and references therein). In addition, oligonucle-
otides of up to 30–50 units could be obtained in abiogenic systems from chemi-
cally activated monomers (e.g. nucleoside 5’-phosphorimidazolides [36]) when
either polynucleotide chains [36-38] or mineral surfaces [39-44] were used as
polymerization templates.
Still, the heterotrophic theory of abiogenesis has encountered certain prob-
lems. Oparin’s initial model implied that primordial atmosphere was reducing,
dominated by methane and hydrogen gas [11,12]. However, according to the
current views, the primordial atmosphere was more oxidized and similar to those
of modern Mars and Venus, where CO2 still makes 95% of the atmosphere with
N2 and H2 being present in small amounts [45-53]. In a CO2-dominated atmo-
sphere, any primordial (bio)chemistry could not start unless CO2 was reduced
to organic molecules capable of participating in pre-biological syntheses (see [16]
and references therein). Straightforward attempts to achieve abiogenic syntheses
of amino acids or nucleobases with CO2-dominated gas mixtures so far proved
unsuccessful [16,54].
The alternative “metabolism first” concept implies that emergence of the first
replicators was preceded by establishment of self-sustaining cycles of chemical re-
actions that could produce increasingly complex organic compounds (see [10,55]
for recent surveys). Currently, there are two detailed evolutionary scenarios repre-
senting the “metabolism first” concept. Wächtershäuser envisioned “two-dimen-
sional” primordial metabolic cycles driven by oxidation of iron monosulfide (FeS)
into iron disulfide (FeS2, pyrite) and confined to the mineral surfaces at the sea
floor [56-61]. Besides the involvement of FeS clusters in a variety of anaerobic en-
zymes, consideration of FeS/FeS2 metabolism had an added benefit of accounting
for the key role of sulfur in cell metabolism. Russell and co-workers [62-71], in
turn, have suggested that the first metabolic cycles started inside porous chimneys
of the deep-sea alkaline hydrothermal vents. It has been suggested that such com-
partmentalized structures consisting of FeS could offer three-dimensional reac-
tion space and provide a framework for the emergence of the first cells [64].
From the viewpoint of energetics, any hypothesis of abiogenesis has to indi-
cate explicitly the energy source(s) that could account for the (i) formation of
reduced carbon compounds and (ii) primordial polymerization reactions. There-
fore, it might be useful to compare the available scenarios of abiogenesis with re-
spect to the underlying energy mechanisms. When considering the field from this
point of view, it transpires that the proponents of the “replication first” scenarios,
and in particular of the RNA World concept, just do not focus on primordial
energetics and leave all options open (see e.g. [16]). Instead, more emphasis is put
on understanding the chemistry of the primeval syntheses and the mechanisms of
information processing in primordial replicating cycles [17-33].
In contrast, proponents of the “metabolism first” concept explicitly address
the energetics problem. Several papers by Wächtershäuser proposed a detailed
chemical mechanism where oxidation of FeS to FeS2 at the sea floor was used to
drive the reduction of either CO2 or CO [56-61]. Indeed, the free energy of the
redox transition of FeS to FeS2, at least under some conditions, is sufficient to
drive the reduction of CO2. Unfortunately, so far, all attempts to yield measur-
able CO2 reduction at the expense of FeS oxidation reaction under simulated
“primordial” conditions have failed (see [72] and references therein). The reason
for this failure, as discussed in detail by Schoonen and co-workers [72], is that for
redox reactions, favorable thermodynamics alone is not sufficient. In addition, the
redox potential of the electron donor has to be lower than that of the electron ac-
ceptor. To drive CO2 reduction at an appreciable rate, one needs a reducing agent
with a redox potential that is lower than the redox potential of the CO2/formate
redox pair, whereas the reducing potential of the FeS/FeS2 redox pair is higher
than that (see Fig. 1). The reduction of CO by FeS is, in principle, possible and
has been reported, albeit at unphysiologically high temperatures [59]. However,
CO is not a major atmospheric constituent and probably never was one. In the
atmospheres of Mars and Venus it is present in trace amounts. Instead, as argued
by Schoonen and co-workers, it could arise in primordial settings predominantly
via CO2 reduction; the reduction of CO2 to CO is, however, even less thermody-
namically favorable than the reduction of CO2 to formic acid [72]. Regarding the
energetics of the primeval polymerization, Wächtershäuser speculated that ionic
binding of the primordial building blocks to the FeS surfaces might facilitate their
interaction and even make the synthetic reactions thermodynamically favorable
[56-58].
Figure 1. Energy diagrams for FeS, ZnS, and MnS as potential donors of photo-excited electrons (left column)
and for the biologically relevant electron acceptors (right column). The Highest Occupied Molecular Orbital
(HOMO) level in the valence bands of each semiconductor is shown by a darker color than the respective
Lowest Unoccupied Molecular Orbital (LUMO) level in the conduction band. The picture is based on data from
references [72,99,122,262,264].
In the most recent of the scenarios put forward by Russell and co-workers,
hydrogen and hydrocarbons were produced below the sea floor in the complex
“serpentinization” reactions and then brought to the surface by hydrothermal flu-
ids (see [71] and references therein). Considering the primeval polymerization
reactions inside the porous, compartmentalized bodies of hydrothermal vents,
these authors suggested that the pH gradient across the inorganic membranes of
these compartments, between the alkaline hydrothermal fluids and the more acid-
ic primordial ocean, could have served as the energy source for primeval syntheses
[70,71], by analogy with the transmembrane proton gradients on the membranes
of modern bacterial cells. However, coupling of the transmembrane proton gradi-
ent to synthetic reactions – even in most primitive bacteria – is performed by a
sophisticated enzyme machinery which seems to be evolutionarily recent [73].
Therefore it remains unclear whether and how such coupling could have occurred
in the inorganic systems.
The Concept of the Photosynthetic Origin of Life in the Zinc

World
A few scholars have invoked the solar radiation as a potential source of energy
for the origin of life (see e.g. [74-85]). However, the idea of driving the abiogen-
esis directly by solar energy has not won much support, despite the Sun being
by far the most powerful energy source on this planet [16,45,86]. The limited
acceptance of this idea is probably due to several factors. First, only short-length
UV quanta carry enough energy to drive primeval organic syntheses in the ab-
sence of enzymes [75]. These quanta were available on the primordial Earth: in
the absence of the ozone shield, the UV component of the solar light was by 2–3
orders of magnitude stronger than now [87,88]. However, the same UV quanta
would cause photo-dissociation of organic compounds. Therefore, most scholars
considered the UV irradiation of the primordial Sun to be a hazard for the first
life forms and suggested searching for life origins at the sea floor (see e.g. [87]).
Second, as the energy of UV quanta could be utilized for the synthetic reactions
in many different ways, there has been no consensus on the particular mecha-
nisms involved. For example, considering the ways of CO2 fixation, Mauzerall
and co-workers advocated the idea of CO2 photo-reduction to formaldehyde in
the presence of dissolved ferrous hydroxide [83,89,90], while other authors have
argued that several naturally occurring minerals possess the properties of broad-
band semiconductors and could perform abiogenic photosynthesis [81,91-93].
Further, since there are several minerals with this capacity, different authors advo-
cated participation of different minerals in the primordial (photo)syntheses. Bard
and co-workers [92,93], and, more recently, Senanayake and Idriss [94] studied
the photosynthesis on the surface of TiO2 (anatase/rutile), Halmann and co-work-
ers tested the CO2 photo-reduction by diverse minerals getting a high outcome
with WO3 (wolframite) [81], while Schoonen and co-workers investigated MnS
(alabandite [95]) and ZnS (sphalerite [96]). Last but not least, no detailed – and
hence testable – scenario of light-driven abiogenesis has been suggested so far.
The first such detailed scenario has been put forward in the accompanying
article [97]. This scenario centers on the role of zinc sulfide (ZnS) as a compound
that uniquely combines several traits that could be decisive for the emergence of
life on Earth. Among others, the arguments included ZnS crystals being, on the
one hand, extremely efficient photo-catalysts capable of reducing CO2 and other
organic compounds with a quantum yield of up to 80% [96,98-103] and, on
the other hand, common constituents of the hydrothermal vent systems typically
coating those that eject fluids with the temperature in the 200 – 300°C range
[104-106]. The Zinc world hypothesis suggests that, as long as the atmospheric
pressure at the surface of primordial Earth remained above ca. 10 bar, porous
ZnS formations could build up in the direct reach of UV-rich sun beams, because
the temperature of liquid water in hot springs could remain above 200°C even
in sub-aerial settings. In this “Zn world”, the energy of light could be used (i)
for the abiogenic, ZnS-mediated photosynthesis of diverse organic compounds,
(ii) for the selection of most photochemically stable of them, and (iii) for driving
the surface-catalyzed polymerization reactions (see [97] for details). This ZnS-
mediated photosynthesis, however, would gradually decline with the drop in at-
mospheric pressure below 10 bar, with the ZnS-coated surfaces persisting only
around deep-sea hydrothermal vents, which continue to extrude hot, Zn-rich flu-
ids up to these days. The hypothesis further envisions that after the submergence
of the Zn-rich formations, sub-aerial biotopes had to cope with the impact of the
generally abundant Fe2+ ions. Iron, unlike zinc, is redox-active, so that the first
life forms had to undergo major changes to adjust to the iron-containing, redox-
active environment.
Approaches to Validation of Evolutionary Hypotheses

Obviously, experimental verification of this (or any other) evolutionary scenario
would prove extremely difficult, if not impossible. However, as noted by Wächter-
shäuser [107], validation of such concepts could be accomplished through rigor-
ous application of Karl Popper’s principles of testing scientific theories [108,109].
These tests include (i) consideration of the experimental evidence, (ii) the relation
of the hypothesis to earlier theories in the field, (iii) falsification tests, and (iv)
demonstration of the explanatory power of the hypothesis. Popper described the
falsification tests as follows: “The purpose of this last kind of test is to find out
how far the new consequences of the theory – whatever may be new in what it
asserts – stand up to the demands of practice, whether raised by purely scientific
experiments, or by practical technological applications. Here too the procedure of
testing turns out to be deductive. With the help of other statements, previously
accepted, certain singular statements – which we may call ‘predictions’ – are de-
duced from the theory; especially predictions that are easily testable or applicable.
From among these statements, those are selected which are not derivable from
the current theory, and more especially those which the current theory contra-
dicts. Next we seek a decision as regards these (and other) derived statements by
comparing them with the results of practical applications and experiments. If this
decision is positive, that is, if the singular conclusions turn out to be acceptable,
or verified, then the theory has, for the time being, passed its test: we have found
no reason to discard it. But if the decision is negative, or in other words, if the
conclusions have been falsified, then their falsification also falsifies the theory
from which they were logically deduced” (quoted from ref. [109]). As noted by
Yarus and colleagues [25], falsification tests are especially important when hy-
potheses cannot be experimentally proven in principle, as in the case of evolution-
ary scenarios. Several falsification tests are then required, since multiple confirmed
predictions would enhance the plausibility of the initial idea [25].
In the accompanying article [97], the available evidence for the Zn world
hypothesis was considered, the underlying physical and chemical mechanisms
were analyzed, and the suggested scenario was compared to other concepts of the
origin of life. Here, in continuation of this analysis, we formulate and check a
set of predictions that follow from the Zn world hypothesis. We also address the
explanatory power of the hypothesis and discuss how the suggested concept could
eventually be experimentally tested.
Results
Falsification Tests of the Zinc World Concept
As noted by Popper, predictions stemming from the tested hypothesis must be
logically uncoupled from the premises on which the hypothesis had been based
[109]. The Zn world hypothesis is based on the three key premises, namely (i) the
experimentally demonstrated ability of ZnS crystals and nanoparticles to photore-
duce CO2 with a high quantum yield [96,99-103,110], (ii) the need of metal-rich
hydrothermal settings for the emergence of first organisms [45,47,62,63,65-67-
,111-114] and the frequent coating of such settings by ZnS [104,115-117], and
(iii) the unique photostablity of polynucleotides that could imply their emergence
in the presence of UV light as a selective factor [33,78,118-121]. Since all these
premises are either physico-chemical or geological, it seemed worthwhile to focus
here on the biological predictions stemming from this hypothesis. A specific trait
of the Zn world would be the constantly elevated concentrations of Zn2+ ions.
Indeed, upon reduction of CO2 by photo-excited electrons, the negative charge
of these electrons is compensated by protons coming from the water [99,122],
according to the equation:
ZnS ∗ + CO2 + 2 H + → HCOOH + Zn 2+ + S (1)
where ZnS* is the photo-excited state of a ZnS crystal, see Fig. 1. The resulting
accumulation of positive charges in the illuminated ZnS crystals leads to their dis-
ruption and to the release of Zn2+ ions, i.e. photo-corrosion. Photo-corrosion can-
not be completely prevented even by applying efficient electron donors (so-called
hole scavengers, see the accompanying article [97] for details and references). It
remains the main obstacle in the practical applications of ZnS in photoelectric
devices, so that the less photo-corrosive TiO2 is routinely used [122,123]. A simi-
lar photo-corrosion should have taken place within the primeval illuminated ZnS
compartments and caused their enrichment in Zn2+ ions. An environment with
continuously elevated Zn2+ content is geochemically unusual. Generally, Zn2+
ions form poorly soluble salts with such widespread anions as phosphate, carbon-
ate and sulfide, which is why the concentration of free Zn2+ in modern seawater
is less than 2 nM [104]. The concentration of Zn2+ ions in primordial anoxic
waters should have been lower than now because of the poor solubility of ZnS, the
predominant Zn2+ source in the ancient ocean [124]. The available estimates of
Zn2+ content in primordial waters are in the range of 10-15-10-12 M [124-126].
Hence, if we could find evidence of the emergence of life in Zn2+-rich habi-
tats, the Zn world hypothesis could be considered confirmed in its key postulate.
Indeed, because the Zn2+ ions should be continuously removed by precipitation,
an elevated Zn2+ content would be expected to persist only at the continuously
photosynthesizing ZnS surfaces, i.e. both the ZnS surfaces and photosynthesis
(as a sink for electrons) are required. Thus, any evidence of the origin of life in a
primeval Zn2+-rich milieu is, at the same time, evidence of ZnS-mediated abio-
genic photosynthesis at the primordial Earth. Such evidence could be obtained
by examining properties of modern prokaryotic and eukaryotic cells: those RNA
and protein molecules that stem from the Zn world could retain certain traits
from their emergence in Zn2+-rich settings owing to the principle of chemistry
conservation. As discussed in more detail elsewhere [8], this principle, which is
implicitly acknowledged by natural scientists, entails that the organismal chem-
istry can retain information about ancient environmental conditions (see e.g.
[45]). Apparently, post-modification of metabolic pathways in response to envi-
ronmental changes is often either not possible or evolutionarily less probable than
simply maintaining the “ancient” intrinsic chemical milieu. For example, the cell
cytoplasm is highly reduced even in those organisms that inhabit oxygenated en-
vironments. The reduced state of the cytoplasm indicates that the first cells have
evolved – and the principal biochemical pathways have been established – before
the atmosphere became oxygenated (which was due to the activity of cyanobacte-
ria at 2 – 2.5 Ga [127]). The principle of chemistry conservation can be applied to
reconstruction of primordial environmental conditions even in those cases when
no reliable geological evidence is available.
The Zn world concept suggests that the environment that housed the first life
forms was enriched in Zn2+ ions. Then the Zn2+ ions, released upon photosyn-
thesis, could interact with the polymers at the ZnS surface. The latter interaction
could be thermodynamically favorable since the polymer molecule could poten-
tially provide several coordinating bonds for a Zn2+ ion (which can form up to 6
of them [128]). However, to use all these bonding modalities, the Zn2+ ion had
either to induce folding of the polymer around itself (see [129]) or to bind several
polymer molecules together. Those RNA and protein molecules that succeeded in
trapping Zn2+ ions, in turn, could get selective advantage either as more stable
or as catalytically active ones. These Zn-containing polymers would then be likely
preserved in the course of evolution and could show up in the modern cells.
Based on these arguments, we have made a set of specific predictions related
to the occurrence of Zn in modern organisms. In the subsequent sections, we test
these predictions one by one. In order to avoid potential biases, we relied, wher-
ever possible, on data extracted from the published literature and the publicly
available databases. This testing turned out to be fairly complicated. Zn2+ ions
are spectroscopically elusive: unlike other biologically-relevant transition metals,
such as Fe, Cu, and Mn, zinc has no characteristic spectral signatures either in op-
tical (UV-visible) or in EPR spectra (see [130] and references therein). Therefore,
analysis of the Zn content of biopolymers cannot rely on spectroscopy, which is,
generally, the method of choice in bioinorganic chemistry. Instead, presence of a
bound Zn2+ ion in a biopolymer has to be revealed either by methods of ana-
lytical chemistry (see e.g. [131,132]), or from structural data, or from functional
measurements, e.g. of the catalytic activity in the presence of different metal ions
(see [133,134] and references therein), or by bioinformatics approaches [135].
We would like to emphasize that the Zn world concept contrasts a variety
of models that center around the role of iron in the emergence of life, either
as Fe2+ ions in solution [77,83,89,90,136], or as iron sulfide [56-58,60-64-
,67,68,71,137,138]. Therefore, while analyzing the data, we specifically looked at
the content of iron and other transition metals that are essential for life (hereafter
“essential metals” [128]).
The Presence of Zn2+ in Modern RNAs

The Zn world scenario implies that the first RNA-like oligomers emerged within
the illuminated ZnS compartments. Then the Zn2+ ions might be preserved in
modern RNA molecules either as structural elements or catalytic cofactors.
Occurrence of Zn the RNA Structures

The idea of modern RNA structures retaining the Zn2+ ions that were trapped by
the first RNA molecules emerging in the zinc-rich habitats leads to the prediction
no. 1: Known RNA structures should be enriched with Zn2+ as compared to other
transition metals. To check this, we have turned to the solved RNA-containing
structures, available in the Protein Data Bank (PDB, [139,140]) and Nucleic Acid
Database [141,142].
A comparison of the transition metal content in the available RNA-contain-
ing structures, as listed in the MERNA (Metals in RNA) database [143,144], is
presented in Table 1 (these data represent transition metal atoms located at the
distance of no more than 6 Å from any atom that is part of the RNA molecule).
RNA-containing structures from the PDB include such essential metals as Zn,
Mn and Co, with Zn seen in 64 structures, more than any other transition metal.
The relatively large number of Co-containing structures is due to the routine use
of cobalt (III) hexamine as a standard stabilizing reagent which mimics hydrated
magnesium [145]. Manganese atoms are seen in 16 structures, whereas no Fe
atoms in the vicinity of RNA molecules have been reported. Our own further
analysis, which used a shorter cut-off of 3 Å, has shown that the majority of these
metal atoms interact not with RNA proper but with the side chains of various
RNA-bound proteins. Still, in some cases we could find transition metal atoms
that interacted directly with nucleotides, namely Zn in the PDB entries 1NLC,
1S03, 1YXP, 1D9F, Mn in the PDB entries 1EHZ, 1N35, 1Y3O, 2G81, and so
on. The certain scarcity of these interactions is due to the poor binding of metal
cations to RNA and, accordingly, the poor selectivity of such binding. Therefore
transition metal atoms are usually seen in those structures that were crystallized
from solutions that contained the respective salts. In the absence of added Zn or
Mn salts, Mg atoms, which are present in standard crystallization media, bind in
the respective positions (as could be judged from the comparative analysis of the
RNA structures that were crystallized several times, with different divalent cations
(see e.g. [146]). Thus, a separate question that, generally, deserves clarification
is the nature of the divalent metal atoms that are bound by the RNA-protein
complexes in vivo. We have tackled this question while trying to clarify the ori-
gin of Cd atoms in the RNA-containing structures. The presence of Cd in 42
RNA-containing structures, as reported in the MERNA database (Table 1), was
intriguing since Cd is not an essential metal. We have checked the Cd-containing
structures and found out that 41 of them represent different ribosomal struc-
tures which were crystallized in the presence of CdCl2 [147,148]; CdCl2 was
apparently used to improve the crystal stability (Dr. Gulnara Yusupova, personal
communication). The remaining Cd-containing structure shows a complex of the
hammerhead ribozyme with substrate RNA that was crystallized in the presence
of 25 mM of CdSO4 [149]. For the 41 structures related to the large ribosomal
subunit, we could tentatively infer the nature of the metal ions that were replaced
by the added Cd2+ ions. Ramakrishnan and co-workers have recently crystallized
the whole bacterial ribosome with bound tRNA and mRNA under physiological
conditions, with Mg2+ as the only divalent ion used upon preparation [150].
This structure contains dozens of Mg2+ ions, a few Zn2+ ions, and no Cd2+ ions.
Apparently the Cd2+ ions in the 41 ribosomal structures occupied the loci that
are normally occupied by either Mg2+ or Zn2+ ions. Since Ramakrishnan and
co-workers crystallized the ribosome without adding Zn salts to the crystalliza-
tion medium [150], the Zn atoms seen in that structure should be the retained
native ones. These data show that, indeed, Zn atoms are found in RNA molecules
and RNA-protein complexes much more often than any other transition metal
atoms.
Table 1. Transition metal content in RNA structures.
Role of Zn in the RNA Catalysis

The metal ions that were trapped by primeval RNA polymers could serve as cata-
lytic centers. This consideration leads to the prediction no. 2: There should be
ribozymes with Zn-dependent catalytic activities.
The detailed catalytic mechanisms of natural ribozymes have been studied
only in few cases (see [35,151-155] for recent comprehensive reviews). RNA
molecules are surrounded by a shell of diverse cations that stabilize the negative
charges of the backbone phosphate groups. Generally, the metal specificity of
ribozymes is low, because of weaker, as compared to proteins, cation binding,
so Mg2+ ions, which are always present in large amounts as stabilizers, can
seemingly occupy any metal-binding site. Therefore, the exact chemical nature
of the catalytically-relevant metal ions is often difficult to determine. In most
cases, the catalytic activity of ribozymes could be restored by several divalent
cations, e.g. by Mg2+, Zn2+, Mn2+, Cd2+, Pb2+. In some cases, however,
only particular ions, e.g. Zn2+ [156,157] or Mn2+ [158,159] were found to be
functional. Taking into account that Cd and Pb do not belong to the essential
metals [128], the available data (see e.g. [35,151-155,160-167]) indicate that
the transition metals that can be relevant for natural RNA catalysis are, in the
first line, Zn and Mn.
Summarizing the RNA-related predictions, it is worth noting that Fe has not
been reported either as a structural constituent of RNA molecules or as a metal
that is important for the RNA catalysis. The striking absence of iron atoms in the
vicinity of natural RNA molecules is likely to be due to the danger of the RNA
cleavage by hydroxyl radicals that could be produced in the presence of redox-
active Fe2+/Fe3+ ions [168-170]. Accordingly, it seems reasonable to suggest that
early evolution of RNA proceeded in habitats that were enriched in Mg, Zn and
Mn, but not in iron.
Traces of the Zn2+-Rich Environment in the Evolutionarily Old

Proteins
Occurrence of Zn the Oldest Protein Folds
The same logics as used above to suggest the possibility of stabilization of the early
RNA folds by photosynthetically released Zn2+ ions is applicable to the first pro-
teins, which, owing to the more versatile chemistry, could bind metal ions even
tighter than RNA molecules do [128]. Zn2+-mediated protein folding has indeed
been reported, see e.g. [171,172]. Hence, it is possible to formulate the prediction
no. 3: Zn2+ ions should be associated with the evolutionarily oldest protein folds.
Yang and colleagues recently checked the distribution of fold superfamilies
(FSF) among 174 complete genomes [173]. Of 1294 FSFs only 49 were reported
to be present in all Archaea, Bacteria and Eukarya with known complete genomes.
It seems reasonable to assume that these FSFs are among the evolutionarily oldest
ones. Table 2 contains data on metals associated with these 49 FSFs [174]. Be-
cause of certain elusiveness of zinc (see above), we have checked the metal content
in those representatives of these 49 FSF whose structures could be found in the
PDB. Zinc atoms were found in representatives of 37 FSFs. Cadmium atoms
were present in the representatives of 21 FSFs. Again, since Cd is not an essential
metal [128], Cd atoms most likely occupy the sites where Mg2+ or Zn2+ ions
are normally bound (see above and [175]). Iron atoms were associated only with
representatives of three FSFs. Manganese has been found in representatives of
19 FSFs, whereas Mg atoms were present in all FSFs except for one. These data
support the idea that formation and stabilization of the oldest protein folds have
occurred in the settings that were rich in Mg, Zn and Mn, but not in Fe.
Occurrence of Zn in the Proteins with the Oldest Functions

The Zn world concept, as well as some other evolutionary scenarios (see [28] and
references therein) imply that the first enzymes could have emerged to overtake
the catalytic functions from ribozymes. An up-to-date list of the known catalytic
activities of ribozymes can be found in ref. [29]. In the absence of proteins or or-
ganic cofactors, ribozymes, especially the artificial ones, can catalyze breakdown
and formation of diverse covalent bonds, as well as group transfer reactions by
employing acid-base catalysis which often proceeds in a metal-assisted way (see
the discussion above and refs. [29,35,151-155,176]). The first enzymes can be ex-
pected to have had similar catalytic activities. If the first enzymes emerged in the
Zn-rich environments, one could expect involvement of Zn2+ ions in the oldest
enzymes, particularly those that catalyze the formation and breakdown of covalent
Table 2. Occurrence of metal atoms in the representatives of the 49 fold superfamilies that are common to
Bacteria, Archaea and Eukarya
Table 2. (Continued)
bonds and the group transfer reactions. This consideration leads to the prediction
no. 4: The enzymes with evolutionarily “oldest” functions, including catalysis of
formation and breakdown of chemical bonds, should depend on zinc.
Zerkle and co-workers recently analyzed biogeochemical signatures, trying to
reconstruct changes in the enzyme metal content in the course of evolution [125].
Their reconstruction showed that 37% of metalloenzymes that could be timed
to the “very early life” were Zn-dependent with their relative fraction dropping
to 19% in modern organisms. The fraction of Mn-dependent enzymes remained
almost constant (10% versus 9%), while the fraction of iron-dependent enzymes
increased from 18% to 34%. These data [125] indicate that presumed evolution-
arily oldest functions were largely performed by Zn-containing enzymes.
To check the prediction on zinc dependence of the enzymes that catalyze for-
mation and breakdown of chemical bonds, as well as group transfer reactions, we
have analyzed the involvement of transition metals as cofactors in different en-

zyme classes according to the enzyme descriptions in the International Union of
Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature, as docu-
mented, among others, in the ExporEnz database [177,178]. Since we needed
specific information on metals as catalysts, and not just as structural elements, we
searched the MACiE (Mechanism, Annotation and Classification in Enzymes)
database [179,180] and its recently described Metal-MACiE supplement, a da-
tabase of metal-based reaction mechanisms [181,182]. Andreini and co-workers
[181] compared metal-containing enzymes listed in these databases with metal-
containing proteins in the PDB and showed that the relative occurrence of cata-
lytic metals in Metal-MACiE matched well that in the PDB. Table 3 shows the
presence of metal cofactors in different groups of enzymes listed in Metal-MACiE
database (complemented by information from the MACiE database to distin-
guish between Fe2+ and Fe3+).
Table 3. Metal dependence of the enzyme catalytic activities.
As follows from Table 3, Fe, Cu and Mo are catalytically active in oxi-

doreductases (which catalyze electron transfer reactions), whereas Zn2+ ions
are the predominant metal cofactors in hydrolases (which catalyze breaking of
chemical bonds with the involvement of a water molecule) and lyases (which
catalyze breaking of various chemical bonds by means other than hydrolysis and
oxidation). For transferases (which catalyze transfer of functional groups from
one molecule to another), the preferred metal cofactor was Mn. Only few hits
were obtained for isomerases, with a preference for Co as a cofactor. As seen in
Table 3, no transition metal hits were obtained for ligases (which catalyze joining
of two molecules by forming a new chemical bond), although, according to the
MACiE database, some ligases depend on Mg2+ ions. Since the number of hits
in the Metal-MACiE database was small for transferases, isomerases and ligases,
the survey of these enzyme classes was expanded by extracting additional data
from BRENDA (BRaunschweig ENzyme DAtabase, [183-185]). BRENDA is
manually curated and contains a wealth of information on the properties of vari-
ous enzymes, including presence of metals and their likely functions [183,184].
In the case of transferases, the involvement of Zn2+ ions as cofactors seemed to
be less specific than of Mn2+; in the vast majority of cases, both Zn2+ and Mn2+
ions could be functionally replaced by other divalent cations such as Mg2+, Ni2+
or Co2+. Involvement of Fe2+ ions in the catalysis by transferases appeared to be
limited to their ability to replace Mg2+, Mn2+, Zn2+, Ni2+ or Co2+ ions; Fe2+
ions were routinely reported to be the least efficient catalysts in the series. The list
of metal-dependent isomerases shows non-specific utilization of several divalent
cations such as Mg2+, Mn2+, Co2+, Zn2+, or Ni2+. In many cases, the highest
enzyme activity, as compared to other cations, was reported with Mn2+ or Co2+
ions, similarly to the data in Table 3. Specific involvement of iron has been shown
only for lysine 2,3-aminomutase, where a FeS redox cluster is involved in electron
exchange with the catalytic site [186]. Ligases generally use divalent cations, such
as Mg2+, Co2+, Zn2+, or Mn2+. No evidence of specific catalytic activity of Fe
in ligases could be obtained from the BRENDA database.
It is noteworthy that only two transition metals, namely Zn and Mn, are
found in the representatives of all six enzyme classes [181]. Zn is by far the most
abundant catalytic transition metal in hydrolases and lyases, whereas Mn seems to
be involved in transferases and, together with Co, in isomerases. In transferases,
isomerases and ligases, the pattern of the transition metal use, with a non-specific
need for a divalent cation as catalyst, resembles the catalytic preferences of ri-
bozymes (see above). The Fe2+ ions, with few exceptions, are not used in catalysis
outside the oxidoreductases.
Altogether, data on metal content of proteins are consistent with the notion
that the early evolution of enzymes could have proceeded in habitats that were
enriched in Mg, Zn, and Mn, but depleted of Fe.
The Elevated Zn2+ Content Inside Modern Cells

If life emerged in the environments that had relatively high levels of Zn2+ ions
[97], the primordial life forms, which lacked the tools to alter their ionic content
[187], should have had high Zn2+ levels as well. In accordance with chemical
continuity principle, the ionic content of the primordial environments should
be conserved inside modern cells. This leads to the prediction no. 5: The total
amount of Zn2+ in the live cell should be elevated as compared to the levels of
other essential transition metals.
Early studies on the total Zn content in several bacteria produced values of
~0.03% of the dry weight, much higher than for any other transition metal except
for Fe [188]. The content of Zn, as compared to Fe, was somewhat smaller in
Escherichia coli, but 2–3 times higher in Micrococcus roseus and Bacillus cereus
[188]. The data for E. coli were recently confirmed by inductively coupled plasma
mass spectrometry analysis of whole-cell lysates, yielding values of ~200 μM for
Zn and 200–300 μM for Fe, depending on the growth conditions [189]. The total
Zn content in the human body tissues is, on average, somewhat higher than that
of Fe, 3–5 mg versus 2.5–5 mg/100 g of tissue (the data were obtained from tissue
samples that had been washed from blood; the amount of other transition metals
was much lower [190]). It is noteworthy that although cells contain comparable
total amounts (100–300 μM) of Fe [191,192] and Zn (see [193] and references
therein), the concentrations of free (labile) ions differ dramatically, with the free
Fe2+/Fe3+ concentration of ~10 μM [191,192] and free Zn2+ present only in
picomolar amounts [130,193]. These data indicate that intracellular Zn levels
are tightly controlled; they also suggest that modern cells are more limited in Zn
than in Fe.
Modern sea water contains somewhat more iron, (about 5 nM of mostly Fe3+)
than Zn2+ (< 2 nM), see [104] and Table 1 in the accompanying article [97].
Hence, compared to the composition of sea water, Zn appears to be the transition
metal that is concentrated to the highest extent in the cell. As noted by Williams
and Fraústo da Silva, the primeval anoxic ocean must have contained Fe2+ ions,
which are more soluble than Fe3+ ions. The available estimates of Fe2+ content
in primordial waters are in the range of 10-6-10-5 M, compared to the estimate
of 10-15-10-12 M for Zn2+ ions [124-126]. Hence, the intracellular Zn concen-
tration of 100–300 μM reflects a very efficient scavenging of Zn2+ ions and is
consistent with the idea that the emergence of first life forms indeed occurred in
very special, Zn2+-rich environments.
The Metallome of the Last Universal Common Ancestor

While the idea that life originated – and the first RNA and protein molecules
evolved – in Zn2+-rich settings appears to be compatible with the available data,
it does not explicitly state whether these Zn2+-rich settings played any direct role
in the formation of the first cells. Although it is hard to make any specific and
verifiable predictions for these matters, in this section we try tracing the possible
roles of Zn2+ ions at the times of the Last Universal Common Ancestor of all living
cellular organisms (LUCA).
All cellular life forms belong to one of the three main branches of the Tree
of Life, Bacteria, Archaea, or Eukarya [194]. The conservation of a set of essen-
tial genes between the three domains of life has been considered as evidence in
favor of the existence of the LUCA, see [195-197] for reviews. Some research-
ers view LUCA as a consortium of replicating entities which shared a common
gene pool [195]. Alternatively, representatives of the LUCA were suggested to be
full-fledged organisms comparable to modern prokaryotes [198,199]. There are
also numerous possible variants between these two extreme visions of the LUCA.
The infrequency of inter-domain transfer of genes responsible for information
processing [200-203] might indicate that these genes, at least at the LUCA stage,
already formed constant genetic cores of the first organisms. At the same time,
the easily spreading metabolic genes could form a common pool of transferable
operational genes [200], such that the organisms, depending on their metabolic
requirements, could acquire the necessary tools from a common gene pool. The
universal conservation of membrane-embedded subunits of the general protein
secretory pathway [204] and the F- and A/V-type ATP synthases [205] has been
considered as an indication that the LUCA was already a membrane-encased life
form [206]. Its membranes, however, had to be permeable to enable the exchange
of genes, proteins and metabolites [73]. The recent modeling by Szathmáry and
co-workers showed that “collective” metabolism, with different replicators con-
tributing different metabolites to the common pool, could be a pre-condition for
the viability of the whole consortium and its resistance to parasites [207].
Koonin and Martin have argued that the LUCA consortia might have dwelled
in networks of iron-sulfur inorganic compartments (“bubbles”) of hydrothermal
chimneys [138]. As discussed in the accompanying article [97], this model fits
nicely into the Zn world concept, provided that the deep-see chimneys built
of FeS are replaced by “spongy” ZnS precipitates encircling the sub-aerial hot
springs. Precipitation of ZnS at the sites of geothermal activity should have led to
continuous formation of new, empty compartments, so that the more competi-
tive consortia could overcome others by “moving in” first. As argued elsewhere
[73,138,208], such a scheme implies an extensive (gene) exchange between the
members of one consortium, but not between dwellers of different, physically dis-
crete inorganic compartments. It therefore resolves a major conundrum between
the notion of extensive gene mixing that is considered a major feature of early
evolution [195] and the requirement of separately evolving units for the Darwin-
ian selection.
To what extent the conclusions on primordial bioinorganic chemistry that we
have drawn from the data on metal content in modern cells could be related to the
LUCA? The intracellular Zn2+ concentration of 10-3–10-4 M is a feature that is
shared by representatives of all there domains [188-190,209]. The simplest way to
explain this remarkable trait is by assuming that the LUCA still lived in Zn-rich
habitats. An alternative explanation would assume that the Zn content of LUCA
was low and then independently increased in all three major lineages, responding
e.g. to the elevation of environmental Zn2+ level from 10-12-10-15 M in the
anoxic ocean up to 10-9 M after its oxygenation [124-126]. The latter possibility,
however, appears unlikely. The high total Zn content in cells is contributed not by
free Zn2+ ions, which are scarce [130,193], but by large number of Zn-binding
proteins mostly involved in processing of RNA and DNA. These proteins are
widespread in all three domains of life and their Zn-binding motifs (in particular,
so called “zinc fingers”) are homologous [210]. Therefore it appears unlikely that
these Zn-binding motifs could independently develop in different lineages. Since
the ligand chemistry of these binding sites is specifically tuned to prefer Zn2+
over other transition metal ions [211,212], it is equally unlikely that they served
first to bind some other metal, e.g. iron, and only later adapted to binding zinc.
It also appears implausible that selective Zn-enrichment of LUCA’s interior could
be accomplished by powerful ion pumps capable of maintaining the huge Zn
gradient between the LUCA’s interior and the surrounding Zn-depleted, anoxic
waters. As argued elsewhere, the LUCA should have had primitive membranes
[73,138,187,206] that could not hold the required Zn concentration gradient of
> 108; even the modern membranes can hardly do that. Thus, the most parsimo-
nious explanation of the high cellular content of Zn in representatives of all three
domains of life is by suggesting that the LUCA thrived in Zn-rich habitats that
apparently were in the ionic equilibrium with the LUCA’s interior.
It is tempting therefore to make prediction no. 6: The proteins that could
be attributed to LUCA should be enriched in Zn. The problem of the LUCA-
specific protein set has been addressed by several authors, see [197] for a review.
After completion of the first microbial genomes, a “minimal” set of genes shared
by these genomes was deduced; it has been speculated that these genes made the
genome of the LUCA [213,214]. With increasing number of sequenced genomes,
the set of genes shared by all genomes kept shrinking; it has become clear that
with just ~50 such genes it would not be possible to build a full-fledged organism
[196,215]. Accordingly, it is now believed that the metabolism of the LUCA was
carried out by operational genes that are not necessarily conserved in all genomes
[216]. However, a small set of genes that are shared by all known genomes is still
believed to form the core of the LUCA’s genome [196,197,215]. The products of
these ubiquitous genes and their metal affinities are listed in Table 4 [217-238]
and show a notable preference for Zn and Mg as metal cofactors. Iron was found
only in some structures of a single protein family (YgjD/Gcp/QRI7) of obscure
function that was originally reported to have O-sialoglycoprotein endopeptidase
activity, later identified as an apurinic endonuclease, and recently shown to be
essential for genome maintenance in Archaea and Eukarya [234,239,240]. These
data indicate that proteins likely to be present in the LUCA – and, hence, the
LUCA itself – existed in a Zn-rich environment. As discussed above, since the
equilibrium Zn concentration in the primordial oceans must have been extremely
low [124-126], a Zn rich environment could persist only due to some steady geo-
chemical reaction leading to continuous release of Zn2+ ions, such as abiogenic
photosynthesis.
Table 4. Association of essential divalent metals and the products of ubiquitous genes.
Summarizing the preceding part of the Results, Zn-rich habitats appear to

have shaped the primeval biochemistry by favoring the emergence of Zn-sta-
bilized protein and RNA folds, as well as Zn-dependent enzymatic reactions.
Seemingly, the development of the first life forms proceeded in the Zn-rich set-
tings up to the stage of the LUCA.
Testing the Explanatory Power of the Zn world concept

The Zn world concept offers an entirely new look at many aspects of the primeval
evolution and biochemistry. Tracking all the facts that could be explained by the
Zn world concept better than by other hypotheses on the origin of life is beyond
the scope of this paper. In particular, we anticipate that this concept will provide
a framework for many observations related to the biochemistry of zinc. Here we
consider only those explanations that clearly separate the suggested concept from
other hypotheses of abiogenesis. Besides, we try focusing on the phenomena that
until now did not have acceptable explanations.
Energetics of Abiogenesis
The Zn world concept explains how both the reduction of CO2 and the primeval
biosyntheses could be driven by solar energy (see the accompanying article [97] for
details). Other hypotheses on the origin of life either do not consider the energetics
of abiogenesis explicitly (heterotrophic origin of life/RNA World) or, as the above
discussed concepts of Wächtershäuser [56-61] and of Russell and co-workers [62-
71], suggest mechanisms that do not seem to be plausible from the physical or
(bio)chemical viewpoints (see [72] and the Background section above).
Photostability of Polynucleotides
As discussed in the accompanying article [97], (poly)nucleotides, especially those
building Watson-Crick pairs, are uniquely photostable (see also [33,120,121]).
The Zn world concept explains this unique photostability by the role of the UV
light not only as an energy source, but also as a selective factor during the first
evolutionary steps. The unique photostability of (poly)nucleotides finds no expla-
nation in any other hypothesis on the origin of life.
The Zinc Paradox

As discussed above, Zn2+ ions are used as cofactors by several groups of enzymes
that are mostly involved in the cleavage or formation of chemical bonds (see Sec-
tion 2.1.2.3). Thereby Zn2+ ions serve as Lewis acids upon catalytic transitions
[128]. Generally, the capacity of a transition metal to serve as a Lewis acid is
determined by its position in the Irving-Williams series and should increase as:
Mn < Fe < Co < Ni < Cu > Zn [241]. Hence Zn, as a Lewis acid, is expected to
be better than Mn or Fe, but worse than Cu. However, as specifically noted by
Williams and Fraústo da Silva [128], the difference between the transition metals
in this respect is not that great, and deviations from the Irving-Williams series are
possible, e.g. owing to the influence of the enzyme ligands. In many experiments,
Zn atoms could be replaced by other transition metal atoms with only minor loss
in the enzyme activity (in some cases, even with an increase in activity) [128,242].
Therefore, the almost exclusive involvement of Zn as cofactor in all these enzymes
has been considered enigmatic, especially taking into account the low levels of Zn
in the seawater [124,128]. Moreover, while prevalence of Zn in certain types of
enzymes could be attributed to the catalytic properties of Zn2+ ions, their ubiqui-
tous involvement as structural elements [128,210,243] had no explanation at all.
This paradoxical prevalence of Zn ions can now be explained by the shaping – and
folding – of first proteins in Zn-rich habitats.
Summarizing this section, we can conclude that the Zn world concept offers
a single parsimonious explanation for a set of diverse observations that have not
been rationalized so far.
Discussion
In this work, we made six non-trivial biological predictions stemming from the
idea of the origin of life in Zn-rich settings. Specifically, we predicted that Zn2+
ions would be preferentially associated with ancient RNA and protein molecules,
including ribozymes and those enzymes that catalyze evolutionarily old reactions.
These predictions were tested using publicly available data, obtained in studies
that had no apparent bias towards Zn. The results of these tests revealed that
modern cells contain surprisingly high levels of Zn, which is mostly bound to its
constituent molecules, DNA, RNA and proteins. The most parsimonious expla-
nation of these observations seems to be that, indeed, the first life forms evolved
in a Zn-rich environment.
In addition, following the Popper’s principles, we have tested the Zn world
concept by considering the ability of this concept to provide explanations for ob-
scure facts that other theories either ignore or cannot explain. The fact that the Zn
world concept has successfully passed all these tests makes it a serious contender
for the title of a syncretic concept of the origin of life.
Zinc World: No Country for Old Iron?

Some of the results obtained in the course of this work were rather unexpected,
for example, the almost complete absence of the Fe atoms in the evolutionarily
oldest protein folds (Table 2) and in putative proteins of the LUCA (Table 4).
The apparent absence of correlation between the supposedly primitive traits of

life forms and the involvement of iron, which could be seen in a variety of tests
(Tables 1, 2, 3, 4), strongly argues against the view that life has emerged in iron-
rich environments [56-58,61-71,77,136]. This iron-centric view is based, among
others, on the fact that the iron-sulfur clusters could serve both as protein cofac-
tors (e.g. in ferredoxins) and crystal units of natural minerals (see [67,244] and
references therein). The argumentation, however, could be equally well applied
to Zn. Zinc atoms and ZnS clusters are prevalent both in hydrothermal settings
[104,117] and, as cofactors, in proteins [128,245-247]. Furthermore, proteins
that coordinate either Zn atoms or ZnS clusters seem to be more widespread than
iron-sulfur proteins [130,245-251]. Ironically, the first zinc-sulfur protein, metal-
lothionein, had been described by Margoshes and Vallee [252] even before the
discovery of the first iron-sulfur protein, ferredoxin [253]. Sequence similarities
between proteins that bind FeS and ZnS clusters were noted e.g. by Williams and
Fraústo da Silva [128]. Some metal-binding protein scaffolds can bind either Fe
or Zn, depending on their relative concentrations (see [254,255] and references
therein). Remarkably, the iron-sulfur cluster assembly protein IscU is capable of
binding Zn2+ ion in its monomeric form [256], whereas three such monomers
have to interact to bind a FeS cluster [257]. The Zn-binding mode could well be
the evolutionarily older one in this protein. While FeS clusters are involved, to a
large extent, in electron transfer reactions (see Table 3), zinc-sulfur proteins are
mostly associated with RNA and DNA, e.g. as zinc fingers [243,245,246,250]. In
the view of the assumed evolutionary primacy of RNA, one could imagine that,
in a Zn-rich environment, zinc-sulfur proteins could have emerged first. In fact, it
is extremely unlikely that FeS clusters could have ever been directly involved with
RNA since they are efficient cleavage agents for both RNA and DNA (see [258]
and references therein), not to mention hazardous hydroxyl radicals that could be
produced in the presence of redox-active Fe2+/Fe3+ ions [168-170]. The Fe atoms
and FeS clusters could replace Zn atoms and ZnS clusters – in some cases – only
after the emergence of enzymes and membranes which could protect RNA and
DNA from the damaging action of iron and its compounds. The redox-active
Fe and Cu atoms could be recruited as redox cofactors (in support to the nu-
cleotide-based cofactors such as NAD(P)H, FMN, FAD, see the accompanying
article [97] and references therein) by enzymes of those energy-converting systems
that eventually replaced the ZnS-mediated photosynthesis. This time pattern is
in agreement with the results of the above discussed analysis of the changes in
biogeochemical signatures through time [125], where the relative fraction of Zn-
dependent enzymes decreased in the course of evolution, whereas the fractions
of the Fe- and Cu-dependent enzymes have increased. The importance of redox
enzymes must have further increased with the oxygenation of Earth habitats, such
that the total content of Fe in modern organisms is compatible with that of Zn.
Zinc World: Metals and First Biotopes

Testing the predictions on metal binding by RNA and protein molecules also re-
vealed a notable presence of Mn atoms in RNA structures and the oldest protein
folds. This presence of Mn might be not accidental. Manganese is unique in at
least two respects:
a) Mn2+ ions are typical constituents of hydrothermal fluids [259,260]. In
experiments that modeled the high-pressure conditions at hydrothermal
vents, MnS precipitated at the same rate as ZnS, i.e. much slower than
sulfides of Fe and Cu [259]. Hence, one can expect that the sulfides of
Zn and Mn could precipitate at approximately the same distance from
the orifices of the primeval sub-aerial hot springs and could form mixed
ZnS/MnS haloes around them, as found in the ancient volcanogenic metal
sulfide (VMS) deposits where the haloes of neighboring vents intersect and
join into networks [105,261].
b) MnS is the only other transition metal sulfide – besides ZnS – that can
photoreduce CO2 albeit, seemingly, with a lower quantum yield (see Fig.
1 and refs. [95,262]). The band gap of MnS is smaller than that of ZnS,
about 3–3.5 eV versus 3.2–3.9 eV (see Fig. 1 and [262-264]). Because of
the smaller band gap, MnS can photoreduce CO2 by using visible light.
Hence, photoactive formations that contained MnS in addition to ZnS could
use for photosynthesis not only the UV quanta, but also the visible light (up to ca.
450 nm), which could increase the productivity of the first photosynthetic com-
munities. In addition, because of its lower scattering, visible light could penetrate
deeper into the porous interior of the photosynthetic edifices. We would like to
note that the possible supportive role of Mn in the Zn world did not follow from
the premises of the original hypothesis [97], but transpired during its testing.
The Zinc world concept provides a plausible answer to the question why some
transition metals are essential for living organisms while others are not. The less
frequent – as compared to Zn and Mn – usage of other transition metals as co-
factors can be explained by their scarcity in the settings that hosted the first life
forms. Generally, hydrothermal fluids contain not only Zn2+, Mn2+ and Fe2+
ions, which we have discussed so far, but also notable amounts of Pb, Cu, Ni,
Co and some other metals, with exact composition varying depending on lo-
cation [104-106,260]. These metals are also found, in variable amounts, in the
ancient VMS deposits (see [261,265] for reviews and the accompanying article
[97] for further details and references). However, the only metal sulfides that can
photoreduce CO2 are ZnS and MnS (see [95,99,122,262,264]). Other transi-
tion metals, if present as substantial impurities in the ZnS/MnS settings, would
function as energy traps for the photo-excited electrons (see Fig. 1 and ref. [264])
and decrease the quantum yield of the abiogenic photosynthesis. Hence, the exact
metal content of sulfide precipitates, most likely, could vary at different spots of
primeval hydrothermal activity depending on (i) the chemical composition of the
underlying crust, (ii) the temperature of hydrothermal fluids and (iii) their pH
value (as it varies nowadays, see [104-106,260]). However, only those precipitates
made of ZnS and MnS could photosynthesize, support the first organisms, and,
hence, be inhabited. Accordingly, if we consider a particular transition metal ion,
the probability of its recruitment for some primeval biochemical task could be
proportional to its concentration at a particular habitat multiplied by the number
of potential “recruiters”, i.e. the life forms present. As a result, some photosyn-
thetically inert transition metals became involved only occasionally (e.g. Co, see
Table 3 and ref. [266]) or upon later evolutionary steps (as Fe, see discussion
above), whereas others failed to attain any essential biological function (e.g. Pb).
The suggested concept might also explain why aluminum, although wide-
spread in the Earth crust and soil, has not been recruited for biochemical tasks.
The sulfides of aluminum, as well as of titanium, are unstable in water. Therefore
aluminum does not precipitate at the spot of hydrothermal activity but becomes
dissolved in water and apparently comes down later, far away from the hydro-
thermal orifices [267]. The absence of aluminum among essential metals, when
combined with the importance of sulfur for biochemistry, appears to discount
those models of abiogenesis that envision the origin of life in clays (see [268] and
references therein) since clays are aluminum silicates that, unlike hydrothermal
sulfide precipitates, do not contain sulfur.
Based on available geochemical data, in particular on the architecture of the an-
cient VMS deposits [105,261,265], one can envision networks of photosynthesiz-
ing and habitable bands of precipitated ZnS and MnS around primeval hot springs.
These networks of joined rings at the spots of geothermal activity, a kind of prime-
val “Yellowstone Park” realm, could represent the first Earth biotopes (see Fig. 2).
Figure 2. A schematic representation of interweaved haloes made of porous ZnS/MnS (shown as aggregates
of grey spheres) around the sub-aerial, hydrothermal hot springs. These networks are proposed to have served
as the Earth’s first biotopes (see the text and the accompanying article [97]). The picture uses data from refs.
[66,105,115,117,260,261].
Decline and Fall of the Zinc World

We can only speculate on the sequence of events that followed the drop in the
atmospheric pressure below 10 bar and the gradual decay in the delivery of hot,
Zn-rich hydrothermal fluids to the illuminated settings. An obvious consequence
of these developments was gradual vanishing of illuminated ZnS surfaces and ces-
sation of abiogenic photosynthesis.
As suggested in the accompanying article [97], ZnS-dependent communi-
ties should have been functionally stratified, just as the modern phototrophic
communities are (see [45,269] and references therein). If so, the inhabitants of
different ZnS strata would encounter vastly different levels of UV irradiation and
evolve under different selective pressures. In particular, those inhabiting upper,
light-exposed layers would need some protection from the damaging UV light.
Such protection could be provided by UV-absorbing porphyrins and/or chlorins
(precursors of chlorophylls). Being attached to proteins, these rings could con-
vert the UV quanta, after absorbing them directly or getting them from nearby
aromatic amino acids, into harmless red quanta [270]. In response to the de-
mise of the ZnS-mediated photosynthesis, the life forms in the upper layers could
use their chlorin-containing proteins to catalyze light-driven separation of elec-
tric charges [270] and thus become capable of reducing such compounds as e.g.
NAD(P)H, which could then convey the electrons to metabolic chains. Sequence
and structural analyses showed that modern photochemical (photosynthetic) re-
action centers could have emerged from dimerization of the ancestral simpler
chlorin-carrying membrane proteins [270-275], which, in turn, could function
as UV-protectors of primordial cells [270]. Halmann and colleagues [81] noted
the similarity between physical mechanisms of the chlorophyll-based and semi-
conductor-based photosyntheses, which both include light-induced charge sepa-
ration followed by the stabilization of the reduced states, as shown in Fig. 3. This
figure also shows that if we focus on the photosynthesis by ZnS nanoparticles,
even the sizes of the abiogenic and biogenic photochemical devices match each
other. In addition, the same reaction of sulfide/sulphur oxidation is used to re-fill
the photo-generated electron vacancies (holes) in the most primitive, homodi-
meric photochemical reaction centers of green sulfur bacteria [276,277]. The dis-
advantage of the modern protein-based photoreaction centers, as compared to
ZnS crystals, is that they cannot reduce CO2 directly. Therefore a full-fledged
protein-based photosynthesis must include some version of the Calvin cycle to
incorporate CO2 into organic molecules at the expense of photoreduced NAD(P)
H. As argued by Gánti [278], the Calvin cycle could develop directly from the
Butlerov’s reaction [279-281], since the sugar intermediates of the Calvin cycle
essentially overlap with the components of this autocatalytic pathway (see also the
accompanying article [97] and references therein). The biogenic photosynthesis
could initially complement the gradually diminishing ZnS-mediated photosyn-

thesis; its contribution, however, should have increased with time, as the forma-
tion of the ZnS surfaces in the illuminated, sub-aerial settings came to the end.
In this framework, the emergence of biogenic photosynthesis might represent a
clear-cut case of functional takeover – with the primeval photochemical reaction
centers and primordial Calvin cycle accomplishing together the function that the
ZnS/MnS covered fields could perform alone, namely the utilization of solar en-
ergy for fixation of CO2 (see Fig. 3).
Figure 3. A comparison of energy diagrams for a photosynthesizing ZnS nanoparticle (left panel, the picture is
taken from the accompanying article [97] and is based on references [98,103,122]) and a bacterial photochemical
reaction center (right panel, a primitive, sulfide-oxidizing reaction center complex of green sulfur bacteria
[276,277] is shown schematically as an example).
Hence, although after the drop in the atmospheric CO2 pressure the photo-
synthesizing ZnS edifices could no longer build up in the illuminated settings, the
life forms could persist in these habitats by relying on the protein-based photosyn-
thesis. In the absence of the ZnS settings, the organisms, however, had to undergo
major changes upon adapting to the new environments. This selective pressure
should have favored formation of encased, bacteria-like entities that could main-
tain – in their interior – the chemical content similar to that in the Zn world, i.e.
the high Zn level needed for the RNA and DNA processing machinery (see the
previous sections). Since the concentration of Zn ions in the sea water is low, these
organisms had to develop active membrane ion pumps to maintain high Zn levels
in their interior, see [282] for reviews.
The Zn world, however, did not vanish completely; fresh, porous ZnS edifices
continued to build up at the sea floor, owing to the high temperature of the deep-
sea hydrothermal fluids. These ZnS habitats could still accommodate life forms,
which, however, could no longer rely on abiogenic photosynthesis. One possible
metabolic strategy for such organisms would be chemoautotrophy, i.e. obtaining
reducing equivalents and energy from oxidation of sulfide or hydrogen, the ap-
proach which they could already practice while thriving in the dark, bottom layers
of the photosynthesizing ZnS settings and which the prokaryotic inhabitants of
hydrothermal vents still use these days. This strategy would impose strict limits
on the size of living organisms, as they would have to maintain a high surface-to-
volume ratio [283]. These organisms could gradually spread away from the ZnS
settings and populate iron-rich settings, provided that they developed the cell
envelopes and other tools to keep the intrinsic Zn level high.
The most conservative survival strategy would be to remain confined to the
ZnS edifices and to retain the ancient heterotrophic way of life, i.e. to consume
organic compounds – e.g. by using Zn-dependent hydrolases [128,284,285] –
that could come with hydrothermal fluids and/or result from the activity of the
chemotrophic organisms. From the evolutionary point of view, such heterotrophs
remained adherent to the primeval way of life and, hence, could retain some an-
cient features (e.g. high dependence of their metabolism on Zn).
The accompanying paper [97] starts with Darwin’s famous notion that emer-
gence of living substance anew is extremely unlikely because “...at the present
day such matter would be instantly devoured or absorbed, which would not have
been the case before living creatures were formed [286]”. Here we argue that the
living matter may have emerged on Earth owing to a unique interplay between
the solar UV-light and the geochemical conditions that brought into existence
the sub-aerial Zn world. Thus, we dare to suggest that once the photosynthesizing
Zn world could not persist anymore – perhaps, partly as a consequence of CO2
consumption by the first life forms – there was no force left to power subsequent
origins of life on Earth.
Separation of the Main Domains of Life

The mechanism of separation of LUCA’s descendants into three main lineages
remains controversial (see [138,287,288] and references therein). Zillig and co-
workers have suggested that the split of Bacteria and Archaea resulted from a geo-
graphic separation of two populations [289]. Gogarten-Boekels and colleagues
proposed a catastrophe, such as a major meteorite impact, with ancestors of Bac-
teria and Archaea, respectively, as survivors of this catastrophe [290]. Woese sug-
gested a ‘genetic annealing’ of the common gene pool as a mechanism leading to
the three domains of life [195]. Martin and co-workers have suggested that Bac-
teria and Archaea are descendants of two distinct populations that thrived within
an iron-sulfur deep-sea hydrothermal vent [64,138].
Whatever the separation mechanism(s), the modern representatives of the
three domains of life are quite different. These differences seem to indicate that
they have evolved, after the splitting of the main lineages, under different environ-
mental conditions. Thus, any scenario of the domain separation has to include a
tentative explanation of the key differences between the three domains of life. For
example, it has to be explained why the (bacterio)chlorophyll-based photosynthe-
sis is found in Bacteria but not in Archaea. Answering this particular question,
Nisbet and Fowler hypothesized that (bacterio)chlorophyll-based photosynthe-
sis has developed, among some inhabitants of the deep-sea hydrothermal vents,
from heat sensors that could react to infrared radiation. These organisms, after
their eventual migration into the sub-aerial habitats, could switch to the photo-
autotrophic growth and eventually give rise to future Bacteria [291]. Alternatively,
Russell and co-workers hypothesized that after the first life emerged at a deep-sea
hydrothermal vent, a geological obduction could bring a portion of the deep-sea
biosphere into the photic zone, with (bacterio)chlorophyll-based photosynthesis
subsequently emerging in this population [65,68].
The Zinc world scenario, in principle, can explain both the emergence of the
main domains of life and the specific traits of the organisms belonging to them.
Indeed, as argued above, the LUCA consortia could have inhabited photosyn-
thesizing, porous ZnS settings. In the previous section, we have discussed the
possibility that the inhabitants of different layers of the ZnS-confined communi-
ties could respond differently to the gradual decay in the ZnS deposition in the
illuminated settings. The inhabitants of the upper layers would be switching to
the (bacterio)chlorophyll-based photosynthesis, whereas the dwellers of the lower,
darker layers would either turn to the chemoautotrophy or, alternatively, become
highly specialized heterotrophs. It is noteworthy that with gradual migration of
the high-temperature hydrothermal systems – and their inhabitants – into the sea
depths, the sub-aerial phototrophic communities would eventually separate from
the consortia staying with the hydrothermal vents. This separation would then
persist at least until the emergence of the swimming mechanisms that enabled
movement in the water column. During this time, discrete lineages would evolve
independently and attain their specific traits.
The outlined hypothetical scenario implies that the demise of ZnS mediated
photosynthesis triggered a major separation of the first life forms into (i) the
sub-aerial communities dependent on (bacterio)chlorophyll-type photosynthesis
as source of reducing equivalents (the future Bacteria) and (ii) the communities
confined to the ZnS settings at the sea floor. The dwellers of the sea floor habitats
could diversify further. Some of their lineages would evolve by developing new
types of metabolism, e.g. chemoautotrophy. Acquisition of cell envelopes would
enable their spread into Zn-poor media and give rise to diverse archaeal branches.
In contrast, the most conservative lineage would remain adherent both to the
ancient ZnS milieu and to the primeval, heterotrophic way of life. Only after the
dwellers of sub-aerial habitats developed swimming machinery they would have

been able to detach from the shoreline and populate the ocean photic zones. A
certain mixing between lineages would then become possible, enabling a “lat-
eral” gene exchange between them [292]. The flow of organic matter from the
surface water layers to the sea floor, owing to the sedimentation processes, had
to be more extensive than in the opposite direction. Due to sedimentation, the
larger, heterotrophic inhabitants of the ZnS settings, not being constrained by
size limitations, could eventually acquire the representatives of phototrophic sub-
aerial communities as endosymbionts. In particular, a symbiosis with respiring
α-proteobacteria, the future mitochondria [293], could, perhaps, rescue some of
these heterotrophs (hereafter pro-eukaryotes) from oxidation and extinction after
the ocean waters became oxygenated [294,295], paving the way to the modern
Eukarya (see [287,288,296] and references therein).
The suggested scenario of the domain separation is based on two premises,
namely (a) that the demise of the ZnS-mediated photosynthesis would have forced
living organisms to search for new sources of energy and (b) that inhabitants of
the stratified ZnS habitats could pursue different strategies upon this search. The
outlined scenario reproduces the actual differences between the representatives of
the major domains and is in agreement with the following observations:
(i) (Bacterio)chlorophyll-based photosynthesis is present in Bacteria but not
in Archaea (see [297] and references therein);
(ii) Inhabitants of the primordial microbial community at the Buck Reef
Chert (a 250- to 400-m thick rock along the South African coast that was
produced by phototrophic microbes ca. 3.4 Ga ago), have been defined as
partially filamentous phototrophs, which apparently used the Calvin cycle
to fix CO2 [298,299]. The reported absence of traces of life in the layers
that corresponded to the deeper (> 200 m) water environments suggests
that 3.4 Ga ago microbial communities of the photic zone were physically
separated from the communities at the sea floor.
(iii) Archaeal metabolic pathways are very diverse; they include heterotrophy
and several different types of chemotrophy [300], in particular, metha-
nogenesis that is specific for Archaea [301]. This diversity suggests that
members of the sea floor communities could have used different survival
strategies and that their exodus from the deep-sea Zn-rich habitats may
have proceeded in several waves. In particular, the strong Ni-dependence
of enzymes that are involved in methanogenesis [301] suggests that this
type of metabolism emerged within habitats that were particularly en-
riched in nickel, for example, in the form of NiS (millerite).
(iv) Cell membranes of Archaea are fundamentally different from that of
Bacteria (see [187,302] and references therein). As argued elsewhere
[73,303], this difference seems to indicate that the formation of modern

ion-tight cell envelopes, needed to survive in chemically hostile environ-
ments, followed the separation of these two domains of life.
(v) Swimming motility mechanisms in different prokaryotic lineages are fun-
damentally different and evolutionarily unrelated [304]; this difference
suggests that the LUCA could not swim. This deficiency, in turn, could
have prevented a major gene exchange between the LUCA’s descendants
dwelling in different environments until the emergence of the first swim-
ming apparata.
(vi) While Archaea contain many operational genes of supposedly bacterial
origin, lateral gene transfer from Archaea to Bacteria was relatively mi-
nor and confined to hyper-thermophilic organisms, such as Thermotoga
maritima [305,306]. This inequality of the lateral gene flows to and from
Bacteria supports the straightforward possibility that the “horisontal”
gene transfer between Bacteria and Archaea/Eukarya predominantly pro-
ceeded downwards relative to the Earth surface, and could be essentially
driven by sedimentation processes.
(vii) No Eukarya-specific autotrophic mechanisms have been reported so far
[64]; their absence might indicate that pro-eukaryotes relied on het-
erotrophy;
(viii) At least 90% of animal biomass of the modern hydrothermal vents de-
pends on the chemoautotrophic endosymbionts (mostly sulfur- and
hydrogen-oxidizing prokaryotes [104,307]). Even single-celled Proto-
zoa at hydrothermal vents exploit prokaryotic symbionts [308]. These
numerous symbioses might reflect a long-lasting cooperation between
the larger heterotrophic inhabitants of the vent communities and the
smaller prokaryotes capable of chemoautotrophy; such cooperation may
have eventually driven the emergence of eukaryotes [293,296].
(ix) The fraction of Zn-containing enzymes in Eukarya is higher than in Bac-
teria or Archaea, see Table 5 and refs. [124,250]. This observation is con-
sistent with the suggestion that pro-eukaryotes remained adherent to the
ZnS settings for a longer time than the ancestors of other lineages.
Table 5. Distribution of zinc-, non-heme iron- and copper-binding proteins in the three domains of life
Several authors who have noted this prevalence of the Zn-dependent enzymes
in eukaryotes (see e.g. [124,250]) attributed it to the evolutionarily recent pro-
liferation of Zn-binding motifs (in particular, zinc fingers) among the Eukarya.
The abundance of the Zn-dependent enzymes in Eukarya [124,250,309,310],
however, is likely to be an ancient feature because it is complemented by the
relative deficiency in the Fe-containing enzymes. The Fe deficiency follows from
the quantitative estimates (see Table 5 and [251,311]), as well as from functional
considerations: eukaryotic cells use the mitochondrial assembly systems to insert
FeS clusters in the apo-proteins of their cytoplasmically and nuclearly located
enzymes [312]. In most cases, an apo-protein is translocated across two mito-
chondrial membranes into the mitochondrial matrix, the FeS cluster is assembled
and inserted, and the folded protein is translocated back into the cytoplasm across
the same two membranes; it is still unclear whether and how the internal mito-
chondrial membrane maintains electric potential of ca. 200 mV while a folded,
FeS cluster-containing protein is being translocated across it. The absence of full-
fledged cytoplasmic machinery for assembling FeS clusters in eukaryotes might
have several explanations. It is possible that the pro-eukaryote possessed the re-
spective enzymes but they were later replaced by the more efficient machinery
of its α-proteobacterial endosymbiont. However, it is hard to imagine that the
(hypothetical) pro-eukaryotic machinery could be even less efficient than the de-
scribed, extremely complicated procedure of inserting FeS clusters into the cyto-
plasmic apo-proteins by the mitochondrial enzymes. In our opinion, it is more
probable that the pro-eukaryote just could not deal with FeS clusters because
it dwelled in Fe-deficient environments. This certain incompetency of the pro-
eukaryote in dealing with Fe follows also from the fact that eukaryotes use the
heme biosynthesis enzymes that are specific for α-proteobacteria and that, most
likely, were acquired from the α-proteobacterial endosymbiont [313]. Therefore
eukaryotes may have colonized Fe-rich habitats later than the representatives of
other domains, i.e. only after a pro-eukaryote entered into a symbiosis with a
respiring α-proteobacterium that provided the host with the Fe-processing en-
zymes. The emergence of respiring α-proteobacteria should, however, follow the
oxygenation of the ocean, at 2.0–2.5 Ga [127]. If so, pro-eukaryotes may have
thrived and evolved in Zn-rich settings for at least 1 Ga, between the separation of
the main domains of life and the oxygenation of biosphere [296,314]. Thus, not
only the LUCA likely dwelled in the Fe-deficient, Zn-rich settings (see above),
pro-eukaryotes may have inhabited these environments as well, and for quite a
long time.
Outlook: Potentially Promising Directions of the

Future Research
Based on the arguments from this paper and the accompanying article [97], we
would like to submit that the Zn world hypothesis has successfully passed the first
set of trials and therefore seems to be worth of further testing. We believe that a
combination of the “bottom up” and “top down” approaches might be decisive
for the further validation of the concept.
Potential “Bottom Up” Trials

The most straightforward “bottom up” trial is to simulate the events in the primor-
dial Zn world. The primordial photosynthesis can be simulated by using porous
precipitates of ZnS and MnS (nano)particles covered by a solution that contains
phosphite and other relevant ions, is saturated with H2 and N2/NH4 + in reason-
able concentrations, and is set under the CO2 pressure of more than 10 bar. After
illumination of this mixture by strong UV light, organic compounds are expected
to form at the photosynthesizing surfaces. Upon simulations, it seems reasonable
to vary the parameters which might affect the yield of photosynthesis, in par-
ticular the relative amounts of MnS, Mg2+, K+, Na+, N2, phosphite, ammonium,
and so on. The tricky task would be determining the exact chemical nature of the
ZnS-adsorbed reaction products; fortunately, some relevant approaches have been
recently developed [43,94], so that this task, hopefully, could be accomplished.
The primordial (photo)chemistry at the surface of a photoactive semiconductor
might dramatically differ from the textbook biochemistry that describes the in-
teractions of chemically stable compounds. It, however, might be inferred from
experimentation. The recent synthesis of activated pyrimidine ribonucleotides
from cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde and inorganic
phosphate in a reaction that bypassed free ribose and the nucleobases serves as a
remarkable example of such an approach [33].
Simulations of the Zn world could also start from other “entry points”. Under
the above described conditions, (photo)polymerization of pre-formed nucleotides
(or nucleosides in the presence of phosphite) could be studied at the ZnS surfaces.
The encouraging results that were obtained upon studying the (photo)polymer-
ization reactions at TiO2 surfaces [94], suggest that nucleotide polymerization at
illuminated ZnS surfaces could proceed with an acceptable quantum yield.
The interactions of pre-formed RNA polymers with ZnS/MnS nanoparticles

(quantum dots, see the accompanying article [97]) and their aggregates also de-
serve investigation. So far, such studies were mostly focused on the interactions
between DNA molecules and CdS nanoparticles (see e.g. [315-318] for reviews
and the accompanying article [97] for further references). Studies of the inter-
actions between RNA molecules and ZnS/MnS nanoparticles under simulated
primeval conditions (see above) could shed light upon the earliest events in the
RNA World. In particular, it seems worthy to check the influence of ZnS surfaces
on the activity of ribozymes.
In addition, the stereoselectivity of the ZnS-mediated photocatalysis (see the
accompanying article [97] and references therein) can be experimentally tested by
applying the already existing approaches [319-323] to the substrates that might
be relevant in the context of abiogenesis.
Since structures of key biological molecules and of ZnS/MnS nanoparticles
are all known, it might be worthwhile to perform a computer modeling of the
interactions between biopolymers at the ZnS (MnS) surfaces. The interactions be-
tween RNA strands, as well as associations of protein chains and RNA molecules
could be modeled at the surface of ZnS templates; the results of such simula-
tions might be of great interest for understanding the ZnS-mediated primeval
syntheses.
Potential “Top-Down” Tasks

A formidable “top-down” task is to reconstruct the tentative biochemistry of the
Zn world using bioinformatics approaches. Comparative genome analysis made it
possible, by searching for the common genes in Bacteria, Archaea and Eukarya, to
reconstruct the gene repertoire that was responsible for the translation and tran-
scription in the LUCA [196,197]. However, this approach did not allow uncover-
ing the metabolism of the LUCA because the metabolic enzymes, owing to the
widespread lateral gene transfer [324], rarely could be definitely attributed to a
particular lineage (as, for example, the F-type membrane ATPases to Bacteria and,
respectively, the A/V-type membrane ATPases to Archaea and Eukarya [205]).
The analysis presented here indicates that the initial steps of evolution proceeded
in the habitats that were rich in Zn, but deficient in Fe. Hence, Fe-dependent en-
zymes were unlikely to be involved in primordial metabolism. With the data from
Table 2 on the oldest protein folds, it seems feasible to reconstruct the initial bio-
chemistry by identifying the metabolic pathways that (i) predominantly involve
Zn- and Mn-dependent, but not Fe-dependent enzymes and (ii) use proteins with
the oldest folds. Leslie Orgel, to whom we would like to dedicate this article, has
noted in his brilliant, posthumously published work [9] that the Calvin cycle differs
from the reverse citric acid cycle in its preferable usage of Mg and Zn as metal
cofactors instead of Fe. Orgel wrote: “It is interesting to compare the kind of
chemistry involved in the Calvin cycle with that involved in the reverse citric acid
cycle. In both cycles, almost all of the molecules involved carry two or more nega-
tive charges. In the Calvin cycle, the great majority of these charges are provided
by phosphate groups, but in the reverse citric acid cycle, carboxylate groups are
the only sources of negative charge. Furthermore, the only reduction that occurs
in the Calvin cycle – the conversion of 3-phosphoglyceric acid to glyceraldehyde-
3-phosphate – occurs via an acylphosphate intermediate. Reduction in the reverse
citric acid cycle never involves a preliminary phosphorylation. Enzymes that use
transition metal ions or iron-sulfur clusters play an important role in the reverse
citric acid cycle, but are absent from the Calvin cycle, which uses Mg2+ and oc-
casionally Zn2+ cofactors in its enzymes. It seems plausible, therefore, that the
enzymes of the reverse citric acid pathway evolved in a region rich in transition
metal ions and sulfur, whereas those of the Calvin cycle evolved where phosphate
and magnesium were abundant. Presumably, one of these two cycles arose before
the other. Is it possible to determine which came first by using information on
biosynthetic pathways and genomics data? A decision on this question, though
not directly relevant to the origin of life, would be of the greatest importance
for understanding the history of protein-based metabolism on the early Earth”
(quoted from ref. [9]). In the framework of the Zn world concept, we can sug-
gest that the Calvin cycle emerged first (see the previous section), while the citric
acid cycle would arise later, concomitant with the Fe-containing electron-transfer
(respiratory) chains. This suggestion agrees with the accepted fact that the amount
of free phosphate in the biosphere has decreased with time (see [8,97,325] and
references therein), so that metabolic cycles based on the phosphate usage, such as
the Calvin cycle, should be evolutionarily older.
Last but not least, life continues to flourish within the ZnS-coated, deep
sea hydrothermal fields, with their inhabitants categorized mostly as Archaea
[115,117]. It might be worthwhile to inspect those ZnS-confined communities
more closely. Although the ocean waters are saturated by oxygen, the interiors of
the chimneys remain anoxic, because of the reduced state of hydrothermal fluids,
so that many inhabitants of the vents are obligatory anaerobes [104]. There is a
small chance that the descendants of pro-eukaryotes might still thrive in the an-
oxic, porous ZnS edifices.
Conclusion
In this article we have validated the Zn world hypothesis by checking the pre-
dictions that followed from it. In addition, we have shown that this hypothesis
explains several observations which so far remained without acceptable rational-

ization. Since the hypothesis has passed all these trials, its key suggestion, namely
that the development of the first life forms could take place within the photosyn-
thesizing ZnS edifices of hydrothermal origin, appears to have been validated.
Further details of the primeval Zn world, and the exact physics and chemistry of
the reactions involved, deserve further clarification.
In the course of this study, we have analyzed the available data on the relative
abundance of transition metals in biological systems. We have found that RNA
molecules and oldest protein folds are associated with Zn and Mn, but not with
Fe. It seems likely therefore that the early evolution proceeded in several distinct
steps, namely (i) the “Zinc Age” with the first replicating entities “grazing” within
photosynthesizing ZnS compartments and evolving into the first proto-cells, (ii)
the “Iron Age”, during which the organisms, after cessation of the ZnS-mediated
photosynthesis, adapted to using abundant, but redox-active iron atoms in their
energy-converting devices, and (iii) the “Oxygen Age” when the increase in the
atmospheric oxygen content has driven the major evolutionary changes aimed at
prevention of the oxidative damage to aerobic organisms. The transitions between
these “ages” probably represent major evolutionary bottlenecks.
While the transition from the anoxic to oxygenated biosphere has been long
recognized as a key evolutionary event (see e.g. [295,297,326,327]), the transition
from the Zinc Age to the Iron Age, as well as the very existence of the primeval Zn
world remained unnoticed until now. It seems likely that the “Lost Zn World” has
not been uncovered earlier because the spectroscopic elusiveness of Zn hindered
the experimental studies of Zn-containing systems. As a result, the importance of
Zn for cell biology has been by and large underestimated (as a notable exception
we would like to acknowledge the contribution of Vallee and co-workers who
focused on Zn-containing enzymes for several decades [248,328]). Only in recent
years, Zn-dependent systems have drawn more attention and the fundamental
role of Zn started to get recognized (see [124,245,246] and references therein).
We hope that this article could contribute to a shift from the Fe-centric inorganic
biochemistry to the Zn-centric one that would better reflect the key role of Zn in
the living nature and its evolution.
The Zn world scenario, by implying that the Zn- and Mn-dependent enzymes
preceded the Fe-dependent ones, offers a new tool – a biochemical time arrow –
for the analyses of the earliest evolutionary events. Up till now, there was no clear
way to arrange cellular systems in the order of their evolutionary emergence (with
the notable exception of the few oxygen-dependent enzymes that have seemingly
replaced the oxygen-independent ones after oxygenation of the atmosphere, see
[327] and references therein). Although some features of the lost Zn world are
reconstructed in this and the accompanying articles, a lot of further work would
be needed to understand the earliest steps of life on the Earth.
Finally, this work suggests that origin of life was not a one-time historical ac-
cident but a natural and, perhaps, potentially inevitable consequence of an inter-
play between the solar UV-light and the geochemical conditions that existed once
on the ancient Earth.
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Evaluation of New Inorganic
Sorbents for Strontium and
Actinide Removal from High-
Level Nuclear Waste Solutions
D. T. Hobbs, M. Nyman, D. G. Medvedev,

A. Tripathi and A. Clearfield
Approximately 130 million liters of high-level nuclear wastes (HLW) are stored
in 49 underground carbon steel tanks at the Savannah River Site (SRS). About
9% (11 million liters) of the waste consists of precipitated metal oxides and hy-
droxides resulting from caustic additions to acidic waste solutions produced from
fuel reprocessing and other operations at the site. The precipitated solids, referred
to as sludge, contain about 60% of the radioactivity and settle to the bottom of
the HLW storage tanks. The remaining volume of HLW is stored as concentrated
liquid and saltcake produced from evaporation of the waste solutions. This frac-
tion of the HLW contains about 40% of the radioactivity and is comprised of
principally 134,137Cs with smaller amounts of 90Sr and alpha-emitting isotopes of
uranium, plutonium, neptunium and other actinide elements.
Processing facilities will disposition this waste by separating the dissolved ra-
dioactive components from the bulk wastes into a small volume fraction followed
by vitrification in the Defense Waste Processing Facility (DWPF). Separation pro-
cesses include settling, decanting the supernate and washing the sludge solids to
reduce the soluble salt content. The washed sludge then transfers into the DWPF.
Operations will retrieve the concentrated liquid and saltcake with the diluted
alkaline salt solution (310 million liters) pretreated in the Salt Waste Processing
Facility (SWPF) or the Actinide Removal Process Facility (ARP) to remove ce-
sium, strontium and alpha-emitting isotopes of plutonium and neptunium. The
separated radioactive components transfer into the DWPF for vitrification with
the sludge fraction of the HLW. The decontaminated liquid waste transfers into
the Saltstone Facility for incorporation into a cement wasteform for onsite dis-
posal as a low-level waste.
The baseline process for 90Sr and actinide removal features batch adsorp-
tion with an inorganic sorbent referred to as monosodium titanate (MST).
The MST contacts alkaline waste solutions diluted to 5.6M in sodium. After
24 hours of contact, crossflow filtration separates the MST containing the
sorbed 90Sr and actinides from the waste solution. The treated waste passes
on to the caustic side solvent extraction process for separation of the 137Cs
from the bulk waste solution. After cesium removal the decontaminated waste
solution passes to the Saltstone facility for disposal. The MST solids and con-
centrated 137Cs fraction transfers to the DWPF for disposal in the borosili-
cate glass wasteform.
Crossflow filtration separates the decontaminated waste solution from the
MST solids containing the sorbed radioactive components. Stainless steel filter
elements planned for use feature nominal 0.1or 0.5-micron pore sizes. This filtra-
tion also captures any entrained undissolved solids associated with the salt solu-
tion retrieved from the high-level waste storage tanks.
Table 1 provides a listing of the current Saltstone waste acceptance criteria
(WAC) for 90Sr and selected alpha-emitting radionuclides. These limits establish
the target concentrations that the process used in the SWPF for 90Sr and actinide
removal must meet. 90Sr removal performance originally served as the chief cri-
terion for selection of MST for use in radiochemical separations at the SRS. With
increased characterization of SRS wastes, actinide removal performance has in-
creased in importance.
Evaluation of New Inorganic Sorbents for Strontium 167
Table 1. Saltstone Waste Acceptance Criteria for Selected Radionuclides
Of the actinides present in SRS waste solutions, plutonium is the most preva-
lent contributor to alpha activity. Testing indicates that plutonium removal by
MST serves as the rate-limiting step that sets the required process cycle time and
equipment footprint in the SWPF. Significant savings in the capital and operating
costs of the SWPF could occur from development of a new sorbent that exhib-
its greater actinide capacity and more rapid removal kinetics than that currently
demonstrated by MST. Even greater cost savings would result if that sorbent re-
moves cesium in addition to 90Sr and actinides. This dual functionality would
reduce or eliminate the need to use the CSSX process for 137Cs resulting in even
further capital and operating cost savings.
Synthesis efforts in this project to date focused on producing a sorbent with
increased 90Sr and actinide removal performance. Specific types of sorbents pro-
duced and evaluated for removal performance include sodium nonatitanate, met-
al-substituted sodium nonatitanates, crystalline silicotitanates, titanosilicates hav-
ing a pharmacosiderite structure and heteropolyniobates. Table 2 provides a list of
sorbent materials tested. Performance testing featured a simulated waste solution
comprised of the major anionic components of SRS waste solutions as the respec-
tive sodium salts and specific amounts of strontium and actinide elements.
Table 2. List of Sorbent Materials Evaluated for Strontium and Actinide Removal
Table 3 provides a summary of the simulated waste solution used to evaluate

new sorbent materials. Testing also featured actual tank waste material diluted to
the same sodium concentration (5.6 M) as that provided by the simulated waste
solution in Table 3. The actual waste solution, however, contains a different salt
composition than that of the simulated waste.
Table 3. Composition of Simulated and Actual Tank Waste Solutions
We evaluated removal performance by contacting a weighed amount of the

sorbent with a measured volume of solution in a shaker bath at 25 ± 2 °C. For tita-
nium-containing sorbents, we add the sorbent to provide the equivalent amount
of titanium as that from 0.4 g/L MST. For non-titanium materials, we added the
sorbent to provide the same number of equivalents as that provided by 0.4 g/L
MST. Typically, we sampled the batch-contact test bottles after 4, 24 and 168
hours and measured solution phase strontium and actinide concentrations after
removing sorbent solids by filtration.
Figures 1 and 2 provide graphs of strontium and plutonium concentrations,
respectively, versus time of contact of the simulated waste solution with selected
sorbent materials. Included in each graph is the performance of the baseline MST
sorbent for comparison. Testing indicates that sodium nonatitanates, pharmaco-
siderite and the heteropolyniobates materials exhibit strong affinity for strontium
and actinides and in some cases higher capacities than MST. Thus, these materials
appear promising candidates for use in treating high-level nuclear waste solutions.
In general crystalline silicotitanate and germanium-substituted pharmacosiderites
exhibited poor actinide removal and, therefore, do not appear to be promising
materials for treating nuclear waste solutions.
Evaluation of New Inorganic Sorbents for Strontium 169
Figure 1. Strontium Removal Performance with Various Sorbents
Figure 2. Plutonium Removal Performance with Various Sorbents
Acknowledgements
We thank the Environmental Molecular Science Program in the Office of Science,
Department of Energy for financial support of this project.
Origin of Selectivity in Tunnel
Type Inorganic Ion Exchangers
Abraham Clearfield, Akhilesh Tripathi, Dmitri Medvedev,

Jose Delgado and May Nyman
The removal of highly radioactive species 137Cs and 90Sr from Weapon’s grade
Tank Waste is a daunting task. The tanks normally are 5-7M in Na+, 1-3M in
NaOH but only ~10-5M in the targeted species. Nevertheless several sorbents and
ion exchangers have been found that are sufficiently selective to be considered
for remediation purposes. We are involved in a collaborative study, joint with
personnel at the Westinghouse Research Center, Sandia National Laboratory and
University of Notre Dame to uncover the origins of this selectivity in these com-
pounds. The presentation will be concerned with the framework titanium silicates
with the sitinikite and pharmacosiderite structures.
Synthetic sitinikite has the ideal formula Na2Ti2O3(SiO4)•2H2O and
was first prepared at Sandia National Laboratory. The crystals are tetragonal a =
7.8082(2), c = 11.9735(4) Å with four formula units per unit cell. The titanium
atoms occur in clusters of four grouped about a 42 axis, two up and two down
rotated by 90°. Each titanium is octahedrally coordinated, sharing edges in such
Origin of Selectivity in Tunnel Type Inorganic Ion Exchangers 171
a way that an inner core or four oxygens and four Ti atoms form a distorted
cubane-like structure1. These cubane-type structures are bridged to each other
through silicate groups along the a- and b-axis directions. The titanium-oxygen
clusters are 7.81 Å apart in both the a- and b-axis directions with the Si atoms at
Z = •, •. In the c-axis direction, the Ti atoms are bridged by oxo-groups. The c-axis
is approximately 12 Å long, which is twice the distance from the center of one
358 cubane-like cluster to its neighbor in the c-axis direction. This arrangement
produces tunnels parallel to the c-axis direction. Perpendicular to the tunnels are
vacancies in the faces or four sides enclosing the tunnel. Half the Na+ are held in
these cavities and the remainder reside in the tunnels. Cs+ can readily exchange
for Na+ within the tunnel but not Na+ in the framework sites. The cesium ions
are eight coordinated at distances from the framework oxygens approximately
equal to the sum of the Cs-O ionic radii.
The Kd values for Cs+ -Na+ exchange are Nuclear Tank Wastes. However, sub-
stitution of 25 mol% Nb(V) for Ti(IV) eliminates half the Na+ from the tunnels.
The Cs+ in the Nb substituted framework forms a twelve coordinate compound2,
with eight framework oxygens and four water molecules. Because of this high co-
ordination number the selectivity for Cs+ is sufficiently enhanced to remove Cs+
from nuclear waste solutions.
In exchange reactions for Sr2+ the reverse is true. The Nb containing phase
forms a seven coordinate complex whereas the non-Nb form gives a ten coordi-
nate Sr2+ complex.
extraordinarily high but fall off to very low values under condi-
tions simulating those inPharmacosiderite is the name of a natural miner-
al of composition K(FeOH)4(PO4)3•H2O. The titanium silicate analog is
K3H(TiO)4(SiO4)3•4H2O. It has the same cluster of four TiO6 octahedra
bridged by silicate groups but it is cubic. As a result it has three intersecting
tunnels perpendicular to each other. Exchange of Cs+ and Sr2+ in these and Ge
substituted forms will be described3.
Acknowledgements
This research was supported by the U.S. Department of Energy’s Environmental
Management Science Program grant no. DE-FG07-01ER6300 with funds sup-
plied through Westinghouse Savannah River Technology Center. Research car-
ried out in part at the National Synchrotron Light Source, Brookhaven National
Laboratory is supported by the U.S. DOE, Division of Materials Sciences and
Division of Chemical Sciences, under contract no. DE-AC02-98CH10886.
References
1. D. M. Poojary, R. A. Cahill and A. Clearfield, Chem. Mater. 6 2364 (1994).
2. A. Tripathi, D. G. Medvedev, M. Nyman and A. Clearfield, J. Solid State Chem.
175, 72 (2003).
3. A. Clearfield, Solid State Sci. 3, 103 (2001).
Development of Inorganic
Membranes for Hydrogen
Separation
Brian L. Bischoff and Roddie R. Judkins
Abstract
This paper presents information and data relative to recent advances in the
development at Oak Ridge National Laboratory of porous inorganic mem-
branes for high-temperature hydrogen separation. The Inorganic Membrane
Technology Laboratory, which was formerly an organizational element
of Bechtel Jacobs Company, LLC, was formally transferred to Oak Ridge
National Laboratory on August 1, 2002, as a result of agreements reached be-
tween Bechtel Jacobs Company, the management and integration contractor
at the East Tennessee Technology Park (formerly the Oak Ridge Gaseous Dif-
fusion Plant or Oak Ridge K-25 Site); UT-Battelle, the management and op-
erating contractor of Oak Ridge National Laboratory; and the U.S. Depart-
ment of Energy (DOE) Oak Ridge Operations Office.
Research emphasis during the last year has been directed toward the develop-
ment of high-permeance (high-flux) and high-separation-factor metal-supported
membranes. Performance data for these membranes are presented and are com-
pared with performance data for membranes previously produced under this
program and for membranes produced by other researchers. New insights into
diffusion mechanisms are included in the discussion. Fifteen products, many of
which are the results of research sponsored by the DOE Fossil Energy Advanced
Research Materials Program, have been declared unclassified and have been ap-
proved for commercial production.
Introduction
Inorganic membranes with pore sizes less than 1 nm offer many advantages over
thin-film palladium membranes and ion-transport membranes for the separation of
hydrogen from a mixed-gas stream. In microporous membranes, the flux is directly
proportional to the pressure, whereas in palladium membranes it is proportional
to the square root of the pressure. Therefore, microporous membranes become the
more attractive option for systems that operate at increased pressure. An added
feature of the microporous membranes is that their permeance increases dramati-
cally with temperature. Consequently, inorganic membranes have the potential to
produce very high fluxes at elevated temperatures and pressures. The membranes
can be fabricated from a variety of materials (ceramics and metals) because the
separation process is purely physical, not ion transport. Proper material selection
can ensure that the membrane will have a long lifetime while maintaining high
flux and selectivity. One further advantage is the relatively low cost of microporous
membranes. Because their fabrication does not require the use of exotic materials
or precious metals, such as palladium, the cost of producing microporous mem-
branes should be low compared with that for palladium membranes.
One disadvantage of microporous inorganic membranes is that they are po-
rous. They can never produce 100% pure gas streams as can thin-film-palladium
or ion-transport membranes. However, when microporous membranes are cou-
pled with pressure swing adsorption (PSA), the combined system can produce
100% hydrogen. In this scenario, PSA would only be required to separate the
final 1% of the impurities, and the coupling of the two technologies should result
in a very compact and efficient separation system.
Membrane Fabrication
The permeance of a homogeneous membrane is inversely proportional to the
membrane thickness. To be effective for gas separations, the mean pore diameter
Development of Inorganic Membranes for Hydrogen Separation 175
should be 2 nm or less. With such small pores, the membrane must be very thin,
preferably less than 2 µm, in order to have the highest flux at the lowest pressure
drop. Such a thin membrane is too weak to support itself and it must be applied
as a layer onto a strong, porous support material, either metal or ceramic. It is
preferable that the separative layer be applied to the inside of the tube for its pro-
tection. Metal is preferred for the support tube for several reasons. For example,
metal tubes are easier to incorporate into a module. Also, ceramic support tubes
can be prone to catastrophic failure. If a tube fails, the broken pieces can result in
a cascading effect, causing others to break.
The primary or separative membrane layer can be applied directly to the sup-
port tube or to an intermediate layer. A layer having an intermediate pore size
applied to the support tube first can provide a better surface for the primary
separative layer, resulting in a thinner and more uniform membrane. The primary
layer should have a mean effective pore diameter of 10 nm or less and preferably
as small as 2 nm. Once the primary layer is in place, various chemical treatments
can be used to reduce the effective pore diameter to the desired value (as low as
0.5 nm).
It is extremely difficult to fabricate a membrane with absolutely no defects.
Fabricated membranes are evaluated by combining measurements made on them
with a model1 to estimate the percentage of flow through the defects and to esti-
mate the amount that the separation factor would be lowered by their presence.
Because a defect can allow the unimpeded flow of both the desired product gas
and the undesired gases, the number of defects must be minimized in order to
achieve a high separation factor. Several methods have been developed to reduce
the effective pore diameter of a defect or to eliminate the defect altogether. These
defect repair methods do not significantly reduce the number of small pores and
thus do not lower the flux rate of hydrogen through the membrane.
Membrane Characterization and Testing

The two most important characteristics of inorganic membranes are permeance
and separation factor. Permeance is a measure of the gas flow rate per unit area
per unit pressure difference. A more fundamental unit is permeability, which is
the permeance multiplied by the thickness of the membrane. In most cases, the
thickness of the membrane is not known very accurately and so permeance is a
more practical unit.
The separation factor is meaningful only with respect to a mixture of two
gases. The ideal separation factor is the ratio of the permeance of the two gases
measured at zero pressure, where there is no interaction or momentum exchange
between them. Each gas flows through the membrane as if the other gas were not
there. The ideal separation factor for a given temperature can be estimated by
measuring the permeance of each gas separately as a function of average pressure
and extrapolating the permeance to zero average pressure. The ideal separation
factor is then the ratio of the zero-pressure permeances.
The transport of gases through membranes behaves differently as the pore
diameter is reduced. Gas transport can also be affected by temperature, and a
change in temperature can affect diffusion differently at different pore diameters.
However, measuring pore diameters that are smaller than 2 nm is extremely dif-
ficult. Therefore, it is critically important to be able to follow the changes in the
transport mechanisms of different gases during pore-diameter reduction to help
determine the extent to which pores have been reduced. A detailed protocol is
followed to help follow the changes in transport mechanisms.
Several theoretically based models have been developed to help understand the
transport mechanisms. One of the most important is the Hard Sphere Model,2,3
which combines the effect of the size of the gas molecule with Knudsen diffu-
sion. Separation by Knudsen diffusion generally treats gas molecules as points
having no molecular dimensions. In reality, the diameter of a pore appears to
the molecule to be the pore diameter minus its own diameter (or its equivalent
hard sphere). Without taking into account the molecular diameter, the separa-
tion factor for free molecule diffusion (Knudsen flow) is the square root of the
molecular weight ratio. With the molecular diameter consideration, the separa-
tion factor for free molecule diffusion (Knudsen flow) is the square root of the
molecular weight ratio (Knudsen separation factor) multiplied by the cube of the
ratio of the difference between the pore diameter and the molecular diameter for
each molecule. The effects that the molecular diameter and molecular size have
on the theoretical separation factor are demonstrated in Figure 1 with several gas
pairs. This model provides a mathematical formula for what is essentially a bridge
between the Knudsen separation factor and the molecular sieve separation factor.
When the pore diameter becomes equal to or less than the larger of the two mol-
ecules, the larger molecule cannot pass through the membrane and the separation
factor becomes infinite (as in a molecular sieve). As can be seen in Figure 1, the
larger the difference in the molecular diameters, the larger the pore diameter can
be where the separation factor becomes infinite, as is the case with hydrogen/CF4
and helium/CF4. The effective hard sphere diameters, in angstroms, of the mol-
ecules used in the calculations for Figure 1, are as follows: helium 2.58, hydrogen
2.97, nitrogen 3.68, carbon dioxide 3.99, carbon tetrafluoride 4.7, and sulfur
dioxide 4.11. The information in Figure 1 clearly shows that there is a potential
for achieving very large separation factors, even at pore diameters larger than the
molecular sieve pore diameter, when there is a difference in the molecular diam-
eters of the gas pair.
Free molecule diffusion is not the only transport mechanism. The next most
important transport mechanism is surface flow. Surface flow occurs when there
is significant adsorption of a gas on the walls of the membrane. While the mol-
ecules are adsorbed on the membrane surfaces, they are in motion and can diffuse
along the surface. In general, the heavier the molecule or the larger the interaction
potential between the membrane surface and the molecule, the larger the adsorp-
tion and the more surface flow occurs. Since this transport mechanism favors the
heavier molecule, it tends to decrease the separation factor. Surface flow has been
included in the full mathematical transport model.3 However, adsorption and
surface flow measurements are required to evaluate constants in the mathematical
formulation. To date, these measurements have only been completed for carbon
dioxide and an alumina membrane at 25°C. Model calculations were then made
for the binary pair (helium and carbon dioxide). Zero surface flow for helium was
assumed. The results of these calculations are shown in Figure 2. As the pore di-
ameter decreases, the gas-phase diffusion decreases and the surface flow increases,
primarily because the amount of surface area increases relative to the pore volume.
This decrease in flow causes the separation factor to decrease until the pore diam-
eter approaches the diameter of carbon dioxide, at which point the transport of
the carbon dioxide decreases sharply while the separation factor increases sharply.
The calculation was based on the flow of the individual pure gases. It does not
take into account the fact that adsorbed carbon dioxide molecules may decrease
the effective size of the pore diameter and may thus impede the flow of the helium
molecules. Therefore, in a mixed-gas separation, the separation factor may be even
smaller than is shown in Figure 2. It should be pointed out that the separation fac-
tor drops below unity and becomes less than one under certain conditions, which
means that the carbon dioxide permeance is larger than the helium permeance.
Figure 1. Separation factors for gas pairs with different relative sizes as a function of pore diameter obtained by
unsing the Hard Sphere Transport Model.
Figure 2. He-CO2 separation factors at 25°C from the Full Transport Model compared with the Hard using the
Hard Sphere Transport Model.
Permeance Measurements
Rapid and highly accurate permeance measurements are the heart and soul of our
membrane development management protocol. Single-point permeance measure-
ments are of little value. Permeance is measured as a function of average pressure.
A linear regression of permeance vs average pressure provides valuable informa-
tion (we use the sum of the feed pressure and permeate pressure, which is twice
the average pressure, and refer to it as Σ P or pressure summation). Initial testing
is performed with air at room temperature. A series of 5 to 25 permeance mea-
surements is made over an average pressure range from about 50 to 200 cm Hg. A
linear regression is calculated, and then calculations are made of zero permeance,
a permeance deviation factor, and the permeance at an average pressure of 75 cm
Hg. The permeance deviation factor is the ratio of the slope of the linear regres-
sion to the zero-pressure permeance. A positive value may indicate viscous flow
from defects in the membrane. These measurements are made on the membrane
at every stage of development.
Membranes that show promise, by having a small permeance deviation factor,
go to the next level of permeance testing, where permeance measurements are
made over the same average pressure range but at more than one temperature,
typically 25, 150, and 250°C. This series of measurements is made with three or
four pure gases selected from helium, hydrogen, oxygen, argon, carbon dioxide,
carbon tetrafluoride, and sulfur hexafluoride. A linear regression with pressure
summation (sum of feed and permeate pressure) is made at each temperature
and for each gas. The ideal separation factor for each gas with respect to helium
is calculated from the zero-pressure permeances. The ideal separation factor is
extrapolated to 1/T = 0. At infinite temperature (1/T = 0), no adsorption would
be expected. Therefore, the flow is primarily free molecule diffusion. The equation
used to calculate the results in Figure 1 can be used with the ideal separation fac-
tor at 1/T = 0 and the molecular diameters to calculate a mean pore diameter for
the membrane. While the accuracy of this pore diameter calculation is unknown,
it does provide a parameter to track the progress in reducing the membrane pore
diameter.
Results
Helium has been found to behave similarly to hydrogen in microporous mem-
branes and is much safer to use in the laboratory. Therefore, most of our pre-
liminary testing has employed helium as a surrogate for hydrogen. Because much
of our testing is completed at temperatures less than 250°C and because sulfur
hexafluoride is more inert than most hydrocarbons or carbon dioxide, sulfur
hexafluoride is often employed to simulate larger hydrocarbons that may be pres-
ent in a gas stream.
Only the membranes that showed promise (i.e. small permeance deviation
factor) in the testing with air at room temperature were subject to testing with
multiple gases at higher temperatures. Results of selected membranes from recent
membrane development work are presented in Tables 1, 2, and 3. Table 1 lists
the permeance of helium, oxygen, carbon dioxide, and sulfur hexafluoride at two
temperatures. The data is listed in reverse chronological order with the most re-
cent work at the top of table and the data at the bottom of the table being from
early in 2002. Ideal separation factors were calculated from the data for each of
the gas pairs (He/O2, He/CO2, and He/SF6) at both temperatures and are pre-
sented in Table 2. Of special note is how much better the most recent membranes
perform. Recent membranes were found to have ideal separation factors of heli-
um from sulfur hexafluoride over 30 at room temperature and over 100 at 250°C.
Work earlier in the year resulted in He/SF6 separation factors mostly in the single
digits and often less than would be expected from Knudsen diffusion. The large
improvement in the separation factor is believed to be attributable to a recent
improvement in the process to eliminate defects. For ideal free molecule diffu-
sion, the ratios of the permeances predict Knudsen separation factors of 3.316 for
He/CO2, 2.827 for He/O2, and 6.041 for He/SF6. An ideal separation factor
greater than this indicates a higher-than-expected separation factor than would
be predicted if Knudsen diffusion alone were the mechanism governing gas flow
through these fine pores.
Table 1 also shows how the permeance consistently increased as the temperature
increased for all gases except the carbon dioxide. Depending on the membrane,
the permeance of carbon dioxide sometimes increased and sometimes decreased
with increasing temperature. This is believed to be a function of the amount of
surface flow occurring along the walls of the pores at room temperature. An in-
crease in permeance with temperature is contrary to what would be predicted if
transport were governed by Knudsen diffusion. This phenomenon is believed to
be caused by a thermally activated diffusion process that is not well understood
at this time. One interesting feature of this mechanism is that it does not seem
to affect all gases in the same way. With the most recent membranes (e.g., 2528b
and 5021b), the permeance of helium increased by a factor of between five and
six when the temperature was increased to 250°C while the permeance of sulfur
hexafluoride only increased by a factor of less than two. It may be possible to take
advantage of this phenomenon, which only appears to occur in very fine pores (or
at least is much more pronounced in fine pores). Adjustment of the temperature
may result in both an increase in hydrogen flux rate and an increase in the separa-
tion factor.
The separation factors extrapolated to 1/T = 0 and the Hard Sphere Model
were used to calculate pore diameter (see Table 3). It is clear from the results
that the Hard Sphere Model does not always accurately describe the transport
of molecules through these small pores. The model does not incorporate surface
diffusion, nor does it account for the increase in permeance that was found when
the temperature was increased. More work will be needed to better understand
these mechanisms so that they can be incorporated into an expanded, more com-
prehensive predictive model.
Table 1. Permeanc data of three gases for a series of membranes at room temperature at and 250°C (scm3/cm2s
cm Hg)
Table 2. Ideal separation factors for He and a second gas at two temperatures
Table 3. Pore diameter of membrane calculated from measured separation factors of helium and each gas and
the Hard Sphere Model (angstroms)
Conclusions
Much of the work during the past year has been directed toward increasing mem-
brane permeance, achieving repeatability with defect-free membranes, and using
materials and techniques that can be approved by the DOE review process and
manufactured on a large scale. Significant progress has been made in all these
areas. We are significantly expanding our understanding of gas transport in inor-
ganic membranes. Recent results have shown ideal separation factors for helium
over sulfur hexafluoride of more than 45 at 23°C and more than 140 at 250°C.
Also, it has been observed that the permeance of helium increases significantly
with increasing temperature. As a result, even higher permeance and separation
factors should be attainable at higher operating temperatures.
Future work will include testing some of the new membranes that have shown
high ideal separation factors for helium over sulfur hexafluoride with hydrogen
to confirm that our results also apply to hydrogen. Also, efforts will be made to
test the best membranes at temperatures approaching 600°C to empirically de-
termine how much the permeance and separation factors increase with increasing
temperature. Finally, the membranes need to be evaluated under simulated coal-
derived synthesis gas conditions to determine their actual separation performance
and long-term stability.
References
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Energy Systems, Inc. Oak Ridge, K-25 Site, Oak Ridge, Tennessee, Octo-
ber 1996.
2. D. E. Fain and G. E. Roettger, “Development of Ceramic Membranes for
Gas Separation,” Proceedings for the Fourth Annual Conference on Fossil
Energy Materials, Oak Ridge, Tennessee, May 15–17, 1990, pp. 183–94.
3. D. E. Fain, G. E. Roettger, and D. E. White, “Development of Ceramic
Membranes for High Temperature Hydrogen Separation,” Proceedings for
the Fifth Annual Conference on Fossil Energy Materials, Oak Ridge, Ten-
nessee, May 14–16, 1991, pp. 55–64.
Nickel (II), Copper (II) and
Zinc (II) Complexes of 9-[2-
(Phosphonomethoxy)ethyl]-
8-azaadenine (9,8aPMEA),
the 8-Aza Derivative of the
Antiviral Nucleotide Analogue
9-[2-(Phosphonomethoxy)
ethyl]adenine (PMEA).
Quantification of Four Isomeric
Species in Aqueous Solution
Raquel B. Gómez-Coca, Antonín Holy, Rosario A. Vilaplana,

Francisco González-Vilchez and Helmut Sigel
Abstract
The acidity constants of the twofold protonated acyclic nucleotide analogue
9-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(9,8aPMEA)+/-, as well as
the stability constants of the M(H;9,8aPMEA)+ and M(9,8aPMEA) com-

plexes with the metal ions M2+ Ni2+, Cu2+ or Zn2+, have been determined by
potentiometric pH titrations in aqueous solution at I 0.1 M(NaNO3) and
25C. The result for the release of the first proton from H2(9,8aPMEA) (pKa
2.73), which originates from the (N1)H+ site, was confirmed by UV-spectro-
photometric measurements. Application of previously determined straight-line
plots of log K M ( R − PO3 ) versus pK HH ( R − PO3 ) for simple phosph(on)ate
M
ligands, R − PO32−, where R represents a residue without an affinity for metal

ions, proves that the primary binding site of 9,8aPMEA2-is the phosphonate
group for all three metal ions studied. By stability constant comparisons with
related ligands it is shown, in agreement with conclusions reached earlier for
the Cu(PMEA) system [PMEA2-=dianion of 9-[2 (phosphonomethoxy)ethyl]
adenine], that in total four different isomers are in equilibrium with each
other, i.e. (i) an open isomer with a sole phosphonate coordination, M(PA)
op
, where PA2-=PMEA2--or 9,8aPMEA2-, (ii) an isomer with a 5-membered
chelate involving the ether oxygen, M(PA)cl/o, (iii) an isomer which con-
tains 5-and7-membered chelates formed by coordination of the phosphonate
group, the etheroxygen and the N3 site of the adenine residue, M(PA)cl/om3,
and finally (iv) a macrochelated isomer involving N7, M(PA)IIv. The CuE+
systems ofPMEA2-and 9,8aPMEA2-behave quite alike; the formation degrees
for Cu(PA)op, CuM(PA)vo, Cu(PA)c/omj and Cu(PA)clm7 are approximately 16,
32, 45 and 7%, respectively, which shows that Cu(PA)clm7 is a minority spe-
cies. In the Ni2+ and ZnE+ systems the open isomer is the dominating one fol-
lowed by M(PA)vo, but there are indications that the other two isomers also
occur to some extent.
Introduction
The acyclic nucleoside phosphonate, 9-[2-(phosphonomethoxy)ethyl]adenine
(PMEA), also known as Adefovir [1], can be considered as an analogue of (2’-
deoxy)adenosine 5’-monophosphate ((d)AMP2-) [2]. PMEA has excellent anti-
viral properties [1] and in the form of its bis(pivaloyloxymethyl)ester, Adefovir
dipivoxil, it has recently been approved by the US Food and Drug Administration
(FDA) for the treatment [3] of hepatitis B patients; these people suffer from an
infection of aDNAvirus.
PMEA and its relatives affect the viral reproduction cycle at the stage ofD-
NA synthesis, i.e., they serve in their diphosphorylated form as substrates for
polymerases and lead after their incorporation to the termination of the growing
nucleic acid chain [1]. Since polymerases depend on the presence of metal ions
[4], we have studied over the past few years the metal ion-binding properties of
Nickel (II), Copper (II) and Zinc (II) 185
PMEA in detail [2,5,6],and suggested also a mechanism [7] which explains why
diphosphorylated PMEA is initially an excellent substrate for nucleic acid poly-
merases [8,9].
The stability determining binding site of PMEA2-is the phosphonate group;
however, biologically important metal ions like Mg2+, Ca2+, Mn2+ and Zn2+
are able to interact also with the ether oxygen atom and this gives rise to the fol-
lowing intramolecular equilibrium (1) [2,5,6]:
Formula 1
This proposed metal ion-ether oxygen interaction is crucial for the suggested
polymerase mechanism [7] which agrees with the observation that deletion of this
etheroxygenora change in its position in the aliphatic chain leads to compounds
which are biologically inactive [8-10].
With certain metal ions like Cu2+ PMEA2-may also undergo an adenine in-
teraction. This adenine interaction occurs for a minority species via N7 [11],
i.e., the phosphonate-coordinated metal ion forms a macrochelate as indicated in
equilibrium (2),
Formula 2
and which is well known to occur in the complexes of AMP,where a phosphate

group is the primary binding site [12,13]. The majority species, however, results
with Cu2+ from an interaction with N3 [2,11,14] in such a way that a M(PMEA)
species, which exists as a fivemembered chelate (eq. (1)), forms in addition a sev-
en-membered chelate involving N3; this species is designated as M(PMEA)cvom3
and consequently, the macrochelated (eq. (2)) and ether oxygen-bound isomers
(eq. (1)) are abbreviated as M(PMEA)cvN7 and M(PMEA)vo, respectively, and.the
open isomer seen in equilibria (1) and (2) as M(PMEA)op. The indicated situation
regarding Cu(PMEA) is most fascinating because for the first time a quantitative
evaluation of a system in which four isomers occur in equilibrium was possible
11].
The relative affinities of N3 versus N7 of an adenine residue are of general
interest since N7 is exposed to the solvent in the major groove of DNA where
as N3 is located in the minor groove[15].Therefore it was desirable to confirm
the observations summarized above for M(PMEA) systems with another acyclic
nucleoside phosphonate. We selected 9-[2-(phosphonomethoxy)ethyl]-8-azaade-
nine (9,8aPMEA) [16], which also exhibits some antiviral activity [17] and which
is shown in its dianionic form together with PMEA2-in Figure 1, and studied
its metal ion-binding properties with Ni2+, Cu2+ and Zn2+. We selected these
metal ions since they are known [18] to have a relatively pronounced affinity
toward N donors. To complete the picture, the previously obtained equilibrium
data [5,11] for the Ni2+ and Zn2+ complexes of PMEA2-were now also evaluated
regarding the equilibrium scheme (3),
Formula 3
where PA2-= PMEA2-or 9,8aPMEA2-. The presented results prove that at least with
Cu2+ all four isomers occur in solution with both ligands, where as with Ni2+ and
Zn2+ the proof of their occurrence is more difficult since the differences in com-
plex stability between the various species are small.
Figure 1. Chemical structures of the dianions of 9-[2-(phosphonomethoxy)ethyl]adenine (= PMEA2- Adefovir)

[1] and of 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine (= 9,8aPMEA2-), together with the structure of PME-
R2-,whereR is a non-interacting residue, and which represents the metal ion-coordinating properties of the ether-
phosphonate chain occurring in PMEA2-and 9,8aPMEA2-. A further ligand to be considered in this study is 9-(4-
phosphonobutyl)adenine, which is abbreviated as dPMEA2-(=3-deoxa-PMEA-)to indicate that its structure
corresponds to that of PMEA2-except that the ether O atom is replaced by a CH2 group.
Materials and Methods

Materials
Twofold protonated 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine, i.e.
H2(9,8aPMEA), was synthesized by alkylation of 8-azaadenine with a synthon
carrying the structural constituents of the required side chain [16]; in fact, the
same lot of compound was used as previously [19]. The aqueous stock solutions
of the ligand were freshly prepared just before the experiments by dissolving the
substance in deionized, ultrapure (MILLI-Q185 PLUS; from Millipore S.A.,
67120 Molsheim, France) CO2-free water, adjusted to pH about 8.5 by adding 2
equivalents of 0.1M NaOH.
The disodium salt of 1,2-diaminoethane-N,N,N’,N’-tetraacetic acid (Na-

zHzEDTA), potassium hydrogen phthaate, HNO3, NaOH (Yitrisol), andtheni-
trate salts of Na+, Ni2+, Cu2+ and Zn+ (all pro analysi) were from Merck AG,
Darmstadt, FRG. All solutions for the potentiometric pH titrations were pre-
pared with ultra pure CO2-free water.The buffer solutions (pH 4.00, 7.00, 9.00
based on the NBS scale; now NIST) used for calibration of the pH-measuring
instruments were from Metrohm AG, Herisau, Switzerland.
The exact concentrations of the stock solutions of the divalent metal ions were
determined by potentiometric pH titrations via their EDTA complexes. The exact
concentration of the ligand solutions was in each experiment newly determined
by the evaluation of the corresponding titration pairs, i.e.the difference in NaOH
consumption between solutions with and without ligand (seeSection 2.3).
Potentiometric pH Titrations
The pH titration curves for the determination of the equilibrium constants in
H20 were recorded with a Metrohm E536 potentiograph connected to a Metro-
hm E665 dosimat and a Metrohm 6.0222.100 combined macro glass electrode.
The pH calibration of the instrument was done with the mentioned buffer solu-
tions at pH 4.00, 7.00 and 9.00. The titer of the NaOH used was determined
with potassium hydrogen phthalate.
The direct pH meter readings were used in the calculmions of the acidity
constants; i.e. these constants determinedatI 0.1M (NaNO3) and 25 Careso-
called practical, mixed or Bronsted constants [20]. They may be converted into
the corresponding concentration constants by subtracting 0.02 from the listed
pKa, values; this conversion term contains both the junction potential of the glass
electrode and the hydrogen ion activity [20,21]. It should be emphasized that the
ionic product of water (Kw) and the mentioned conversion term do not enter into
our calculation procedures because we always evaluated the differences in NaOH
consumption between a pair of solutions, i.e. with and without ligand. The stabil-
ity constants determined are, as usual, concentration constants.
All equilibrium constants were calculated by curve-fitting procedures in the
way and with the equipment described recently [11, 22].
Determination of Equilibrium Constants

H H
The acidity constants ( K H 2 ( 9,8 aPMEA) ) and K H ( 9,8 aPMEA) of H2(9,8aPMEA)± (see
eqs (4) and (5)), where one proton is at the nucleobase moiety and the other at
the phosphonate group, were determined by titrating 30 mL of aqueous 2.3-2-
.5mM HNO3 (25°C; 1=0.1M, NaNO3) in the presence and absence of 0.4 mM
deprotonated ligand under N2 with 2.2-2.5 mL of 0.03 M NaOH. The differ-
ences in NaOH consumption between such a pair of titrations were used for
the calculations. The pH ranges evaluated were 2.8-8.6 and 3.4-7.8. Under these
experimental conditions the initial formation degree of H2(9,8aPMEA) ± is about
46% and 18%, respectively, and at the end of the titration about 2% and 10% of
H(9,8aPMEA)- are left, respectively. The results for the acidity constants are the
averages of 15 pairs of independent titrations.
M M
The stability constants K M ( H ;9,8 aPMEA) and K M (9,8 aPMEA) of M(H;9,8aPMEA)+
and M(9,8aPMEA) (eqs (6) and (7)), were determined under the same condi-
tions as the acidity constants but now the HNO3 concentration was reduced to
0.83 mM and hence, only mL of 0.03 NaOH was needed for a titration. NaNO3
was partly replaced by M (NO3)2 (25°C; I=0.1 M). The M2+/ligand ratios were
for Cu2+ 11:1 and 5.5:1, for Ni2+ 50:1 and 25:1, and for Zn2+ 28:1, 26.5:1
and 11:1.
The stability constants were calculated [23] by considering the species H+,
H2(9,8aPMEA)+, H(9,SaPMEA)-, 9,SaPMEA2-, M2+, M(H;9,SaPMEA)+
and M(9,8aPMEA). The experimental data were collected every 0.1 pH unit
from about 4% (Ni2+), 1.6% (Cu2+) and 2.4% (Zn+) complex formation of
M(H;9,8aPMEA)+ to a neutralization degree of about 90% with respect to the
species H(9,8aPMEA)-, or until the beginning of the hydrolysis of M(aq)2+,
which was evident from the titrations without ligand. The maximal formation
degrees for the Ni(H;9,8aPMEA)+, Cu(H;9,8aPMEA)+ and Zn(H;9,8aPMEA)+
complexes were only 8.7%, 3.3% and 6.3%, respectively, and hence, the sta-
bility constants of the monoprotonated M(H;9,8aPMEA)+ species are estimates
only. For the Ni(9,8aPMEA), Cu(9,8aPMEA) and Zn(9,8aPMEA) complexes
the maximal formation degree reached in the experiments was about 71%, 51%,
and 18%, respectively; the reason for the low formation degree of Zn(9,8aPMEA)
is that the experiments were hampered by precipitation.
The individual results for the stability constants showed no dependence on
pH or on the excess of metal ion concentration used. The results are in each case
the averages of at least 5 independent pairs of titration curves.
Spectrophotometric Measurements
The acidity constant that describes the release of the proton from the (Nl)H+
site of the adenine residue in H2(9,8aPMEA)+, pK HH2 (9,8 aPMEA) (eq (4)), was also
determined by spectrophotometry. The UV-Vis spectra of 9,8aPMEA (1.2mM)
were recorded in aqueous solution (25°C; I=0.1 M, NaCI) and l-cm quartz cells
with a Varian Cary 3C spectrophotometer connected to an IBM-compatible desk

computer (OS/2 system) and an EPSON Stylus 1500 printer. The pH of the so-
lutions was adjusted by dotting with relatively concentrated HC1 and measured
with a Metrohm 713 pH meter using a Metrohm 6.204.100 glass electrode. The
spectra were recorded within the range of 205 to 330 nm; for further details see
Figures 2 and 3 in Section 3.1.
Results and Discussion

Derivatives of purines are well known to undergo self-association via π-stacking
[24]. Therefore, all potentiometric pH titrations (25°C; I 0.1 M, NaNO3), the re-
sults of which are summarized below, were carried out with a ligand concentration
of 0.4 mM. Under these conditions self-stacking is negligibly small as has been
shown for PMEA [5]. Hence, it is ascertained that the results given below reflect
the properties of monomeric species.
Acidity Constants of H2(9,8aPMEA)±

From the structure of 9,8aPMEA2- (seeFigure l) it is evident that this species can
accept three protons, two at the phosphonate group and one at the N1 site of the
8-azaadenine residue [25,26]. Further protonations at an adeniner esidue are pos-
sible at N7 and N3, but these protons are released with pKa<0 [27]; similarly, re-
lease of the first proton from the -P(O)(OH)2 group of H3(PMEA)+ occurs with
pKa 1.2[26,28]and the same may be surmised for H3(9,8aPMEA). Hence, in the
present study, for which all potentiometric pH titrations were carried out at pH
> 2.8, only the following two deprotonation reactions, in which 9,8aPMEA2-and
related species like PMEA2-(Figure 1) are abbreviated as PA2-(this also holds for
other equations further below), need to be considered:
H 2 ( PA )  H ( PA ) + H +
± −
(4a)
K H
=  H ( PA)   H  /  H 2 ( PA) ± 
− +
H 2 ( PA )
(4b)

H ( PA) −  PA2− + H + (5a)
K HH( PA) =  PA2−   H +  /  H ( PA) − 
(5b)
Indeed, all the experimental data from the potentiometric pH titrations in
aqueous solution could be excellently fitted by taking into account equilibria (4)
and (5). The acidity constants obtained in the present study for H2(9,8aPMEA)±
are given in Table together with some related data [29-31].
From a quick comparison of the acidity constants in Table 1 it is immediately

evident that the first proton released from H2(9,8aPMEA) ± according to equi-
librium (4) is from the (N1)H site and the second one according to equilibrium
(5) from the -P(O)2(OH)- group. This site attribution is confirmed by the spec-
trophotometric measurements seen in Figure 2; the change in absorption of the
H2(9,8aPMEA) ±/
Table 1. Negative Logarithms of the Acidity Constants of H2(9,8aPMEA) ± and H2(PMEA) ± (eqs (4)and (5)),
as Determined by Potentiometric pH Titrations in Aqueous Solution (25°C; I=0.1 M, NaNO3), Together with
Some Further Related Dataa
The error limits given are three times the standard error of the mean value or the sum of the probable
a.
systematic errors, whichever is larger. So-called practical (or mixed) acidity constants are listed; see Section
2.2.
Determined by 1H-NMR shift [25] and spectrophotometric [29] measurements, respectively; 9MeSazaAde
b.
9-methyl-8-azaadenine.
The result pK HH (9,8aPMEA) = 2.73 ± 0.02 was confirmed by spectrophotometric measurements
c. 2
(see Figures 2 and 3); pK HH (9,8 aPMEA) = 2.73 ± 0.08a

2
Average value from compounds like R-CH2CH2-O-CH2-P(O)2(OH)-, where R =H or cytosine (bound via
d.
Nl); for details see ref. [30].
H(9,8aPMEA)- pair occurs in this range of wavelengths where protonation/

deprotonation reactions of related aromatic moieties are commonly seen [32].
A further reason for the spectrophotometric measurements was that the for-
mation degree of the H2(9,8aPMEA) ±species that could be reached in the poten-
tiometric pH titrations was relatively low (see Section 2.3). This means that it was
desirable to determine the acidity constant for equilibrium (4) also by another in-
dependent method. Therefore we measured the absorption spectra of 9,8aPMEA
asafunctionof pH; a representative set of spectra is shown in Figure 2. The evalu-
ation of the same experiment by a curve-fitting procedure, but involving more
data, is given in Figure 3. Since NaNO3 absorbs in part of the wavelength range
needed for the evaluation of 9,8aPMEA data, I was now adjusted to 0.1M with
NaCI. The H 2.73 + 0.08, and this value is final result from two independent
series of measurements is pK HH (9,8 aPMEA) = 2.73 ± 0.08 in excellent agreement with
2
the constant given in Table and determined by potentiometry.

Figure 2. UV absorption spectra measured in 1-cm quartz cells of 9,8aPMEA (1.2mM) in aqueous solution
in dependence on pH; i.e., the pH values varied from 1.207, 2.286, 2.525, 2.796, 3.047, 3.841 to 5.03 I. The
sample beam contained 9,SaPMEA, HCI and NaCI, and the reference beam HC1 and NaCl (25°C; I=0. M,
NaCI). For the evaluation of the spectra see Figure3.
Figure 3. The UV absorption spectra of 9,SaPMEA (Figure2) in aqueous solution were evaluated at 210, 240,
260, 280 and 290 nm in dependence on pH. These evaluations furnished only the first acidity constant of
H2(9,8aPMEA)+. Giving the averaged result (weighted mean) pK HH2 (9,8 aPMEA) = 2.67 ± 0.10 (3s ) for this
experiment (25 C; 1 0.1 M, NaCI). The solid curves shown are the computer calculated best fits for the various
wavelengths through the experimental data points obtained at pH 1.082, 1.207, 1.294, 1.389, 1.719, 1.881,
2.095, 2.286, 2.525, 2.712, 2.796, 3.047, 3.432, 3.788, 3.841, 4.291, 4.811, 5.031, 5.331 and 5.436 (from
left to right) by using the mentioned average of the acidity constant. The seven solid (*) points, i.e., at pH 1.207,
2.286, 2.525, 2.796, 3.047, 3.841 and 5.031 are those that correspond to the spectra shown in Figure 2. The
final result ( pK H = 2.73 ± 0.08(3s )) is the averag eof two independent experimental series.
H 2 (9,8 aPMEA )
The most obvious conclusions from the data in Table 1 are that replacement of
(C8)H by a nitrogen atom reduces the pKa, of the (N1)H+ site by about ∆pKa,
1.5, i.e., this site becomes considerably more acidic as follows from a compari-
son of entries and 2 with 3 and 5. In contrast, entries 2-4 demonstrate that the
nucleobase residue hardly affects the release of the proton from the -P(O)2(OH)-
group. However, elimination of the ether oxygen from the R-CH2CH2 -O-
2− 2−
CH2 -PO 3 -chain enhances the basicity of the -PO 3 -group remarkably
(cf. entries 2-6).
Stability Constants of the M(H;9,8aPMEA)+ and M(9,8aPMEA)

Complexes
Since under the experimental conditions the metal ions (M2+) are present in a
large excess compared to the concentration of the ligand only the following two
equilibria need to be considered for complex formation:
M 2+ + H ( PA )  M ( H ; PA )
− +
(6a)
 (  )
K MM( H ; PA) =  M ( H ; PA )  /  M 2+   H ( PA ) 
+ −
 (6b)
M 2+ + PA2−  M ( PA) (7a)

(
K MM ( PA) = [ M ( PA) ] /  M 2+   PA2− ) (7b)
It should be noted that in formulas like M(H;PA)+ the H+ and PA2-are sepa-
rated by a semicolon to facilitate reading, yet they appear within the same paren-
theses to indicate that the proton is at the ligand without defining its location.
Indeed, together with equilibria (4) and (5), equilibria (6) and (7) are suffi-
cient to obtain excellent fitting of the titration data (see Section 2.3), provided the
evaluation is not carried into the pH range where formation of hydroxo species
occurs, which was evident from the titrations without ligand. Of course, equilib-
ria (6) and (7) are also connected via equilibrium (8)
M ( H ; PA) +  M ( PA) + H + (8a)
K MH ( H ; PA) = [ M ( PA) ]  H +  /  M ( H ; PA) + 

(8b)
and the corresponding acidity constant (eq. (8b)) may be calculated with equa-
tion (9) [33]:
pK MH ( H ; PA) = pK HH( PA) + log K MM ( H ; PA) − log K MM ( PA) (9)

The results are listed in column 4 of Table 2 together with the constants for the
corresponding M(PMEA) complexes and some further related data. The stability
constants given in footnote “e” for the M(H;9,8aPMEA)+complexesneed tobe
considered as estimates since the formation degree ofthese species was low (see
Section 2.3). The stability constants of the M(9,8aPMEA) complexes show the
trend expected for divalent 3d metal ions, i.e., they vary within the series Ni2+
< Cu2+ > Zn2+, and this holds for the constants due to the M(H;9,8aPMEA)+
species as well.
The analysis of potentiometric pH titrations only yields the amount and distri-
bution of the species of a net charged type; i.e., further information is required to
locate the binding sites of the proton and the metal ion in the M(H;9,8aPMEA)+
species. At first one may ask where the proton is located because binding of a
metal ion to a protonated ligand commonly leads to an acidification of the ligand-
bound proton [34,35]. Hence, the acidity constants according to equilibrium (8)
are needed; these values are calculated with the data listed in Tables 1 and 2 by
application of equation (9) to give the following results:
pK NiH ( H ;9,8 aPMEA) = 5.30 ± 0.26 (10a)
( H ;9,8 aPMEA ) = 3.82 ± 0.25 (10b)

H
pK Cu
( H ;9,8 aPMEA ) = 4.83 ± 0.27 (10c)

H
pK Zn
It is revealing to see that these acidity constants of the M(H;9,8aPMEA)+

complexes are by about 1.5 to 3.0 log units smaller than pK H
= 6.85 ± 0.02
H 2 ( H ;9,8 aPMEA )
(Table 1) but approximately 1.1 to 2.6 log units larger than pK H

H ( H ;9,8
2 aPMEA ) = 2.73 ± 0.02
(Table 1). This comparison shows that the proton in M(H;9,8aPMEA)+ is bound
to the phosphonate group, hence, one may tentatively assume that the metal ion
is coordinated preferentially to the nucleobase, since a monoprotonated phos-
phonate group is only a weak binding site. Indeed, this suggestion agrees with
evidence obtained previously for other related M(H;PA)+ species [5,14,36].
Evaluation of the Stabilities of the M(9,8aPMEA)Complexes

For the M(9,8aPMEA) complexes the question arises: Does the 8-azaadenine resi-
due also participate in metal ion binding next to the phosphonate group? Should
such an additional interaction with the nucleobase residue occur then it has to be
reflected in an increased complex stability [37]. Hence, it is necessary to define
2−
the stability of a pure -PO 3 /M2+ interaction.This can be done by applying the
previously defined [5] straight-line correlations which are based on log K MM ( R − PO ) 3
versus pK HH( R − PO ) plots for simple phosphate monoesters [38] and phosphonates
2−
3
[5]; these ligands are abbreviated as R-PO 3 , where R represents a noncoordinat-

ing residue. The parameters for the corresponding straight-line equations, which
are defined by equation (11),
log K MM ( R − PO3 ) = m ⋅ pK HH( R − PO3 ) + b (11)
have been tabulated [2a,5,39,40], i.e.,the slopes m and the intercepts b with the
y-axis. Hence, with a known pKa value for the deprotonation of a-P(O)2(OH)-
group an expected stability constant can be calculated for any phosph(on)ate-
metal ion complex.
The plots of log K MM ( R − PO ) versus pK HH( R − PO ) according to equation (11) are shown
3 3
in Figure 4 for the 1:1 complexes of Cu2+ and Zn2+, as examples, with the data
points (empty circles) of the eight simple ligand systems used [5] for the determi-
nation of the straight baselines. The two solid circles refer to the corresponding
M(9,8aPMEA) complexes and the crossed ones to the M(PMEA) species. For
further comparison also the data points for the related M(PME-R) (solid squares)
and M(dPMEA) (empty squares) systems are shown.
All the latter mentioned data points are clearly positioned above their refer-
2−
ence lines thus proving that beyond the -PO 3 /M2+ binding additional in-
teractions occur. The smallest stability increase is observed for the M(dPMEA)
complexes, where dPMEA2-=3’-deoxa-PMEA2- (i.e.,the ether O is replaced
by CH2) 9(4-phosphonobutyl) adenine (Figure 1); in these instances mac-
rochelates according to equilibrium (2) involving N7 ofthe adenine residue
are formed 11]. For the M(PME-R) complexes the stability increase is more
pronounced and clearly attributable to equilibrium (1) since no other addi-
tional binding site but the ether O atom is available (Figure 1) [5,30]. How-
ever, the stability increase observed for the Cu(9,8aPMEA), Cu(PMEA) and
Zn(9,8aPMEA) species is much larger than the one for the M(dPMEA) and
M(PME-R) complexes, thus indicating that an accumulation of extra inter-
actions occurs as it is depicted in the .equilibrium scheme (3). No meaning
should be attributed to the apparent equality of the stability increase seen in
Figure 4 for the Zn(PMEA) and Zn(PME-R) complexes because the stability
constant for Zn(PMEA) is only an estimate carrying a large error limit (see
Table 2, entry c in column 4).
Extent of the Total Amount of Chelates Formed in the M(PA)

Systems
Before considering the situation in the M(PMEA) and M(9,8aPMEA) complexes
according to the equilibrium scheme (3) in more detail (see Section 3.5), it is
appropriate to evaluate first the total amount of closed species, M(PA)evtot, for all
four PA2-ligands considered (Figure 1) because evidently the sum of all the closed
species, independent of their structure, is responsible for the observed stability
increase. Stability enhancements like those seen in Figure 4 can be quantified by
the differences between the experimentally (exptl) measured stability constants
and those calculated (calcd) according to equation (11); this difference is defined
in equation (12),
log ∆ M / PA = log K MM ( PA)exp tl − log K MM ( PA)calcd
(12a,b)
= log K MM ( PA) − log K MM ( PA)op
M M
where the expressions log K M ( PA)calcd and log K M ( PA)op are synonymous because the
calculated value equals the stability constant, of the ’open’ isomer, M(PA)op (see
2−
equilibria (1)-(3)), in which only a -PO 3 /M2+/ interactionoccurs.In columns
4-6 of Table 2 the values for the terms of equation (12) are listed.
Figure 4. Evidence for an enhanced stability of the M(PMEA) ((⊗)) and M(9,8aPMEA)(•) complexes of
Cu2+ and Zn2+ in comparison with the stability of the corresponding complexes formed with PME-R2-(♦) and
M
dPMEA2-(◊) (for the structures of the PA2-ligands see Figure 1), based on the relationship between log K M ( R − PO3 )
H
versus pK H ( R − PO3 ) for M(R-PO3) complexes of simple phosphate monoester and phosphonate ligands
2−
(R-PO 3 ) (O): 4-nitrophenyl phosphate (NPhp2-), phenyl phosphate (php2-), uridine 5’-monophosphate
(UMp2-), D-ribose 5-monophosphate (RibMp2-), thymidine [-l-(2-deoxy-13-D-ribofuranosyl)thymine] 5’-
monophosphate (dTMP2-), n-butyl phosphate (Bup2-), methanephosphonate (MeP2-) and ethanephosphonate
(EtP2-) (from left to right). The least-squares lines (eq. (11)) are drawn through the corresponding 8 data sets
(O) taken from ref. [38] for the phosphate monoesters and from ref. [5] for the phosphonates. The points due
to the equilibrium constants for the M2+/PA2-systems are based on the values listed in Tables (column 4) and
2 (columns 4 or 6). The vertical broken lines emphasize the stability differences from the reference lines; they
equal log ∆ M / PA as defined in eq. (12) for the M(PA) complexes. All the plotted equilibrium constants refer to
aqueous solutions at 25°C andI=0.1M (NaNO3).
All values for log ∆ M / PA are positive with the single exception of the one for
the Zn(dPMEA) complex where log ∆ Zn / dPMEA is zero within the error limits
(Table 2, entry 4c in column 6).
The ‘total’ of the dimensionless intramolecular equilibrium constant, Kl/tot, is
defined by equation (13) (see also below eq. (21)),
Kl/tot= [M(PA)cl/tot/][M(PA)op] (13)
and values for Kl/tot can be calculated following known procedures [5,12,37,39,40],
i.e.,via equation (14):
K l / tot = 10log ∆ M / PA − 1 (14)

Knowledge of Kl/tot allows then according to equation (15)
%M(PA)cl/tot = 100•Kl/tot](1+Kl/tot) (15)
to obtain the percentage of the sum of all the closed isomers (cl/tot) present in
equilibrium, i.e., their total formation degree. The corresponding results for the
four PA2-ligands of Figure and their Ni2+, Cu2+ and Zn2+ complexes are summa-
rized in columns 6-8 of Table 2.
The most easily understood result ofthe evaluation is the one given under
entry 3 in Table 2 because the PME-R2- ligand can only form the two isomeric
complexes seen in equilibrium (1), i.e. here only the open species, M(PA)op, and
the ether oxygen-closed one, M(PA)cl/O, exist and therefore in these cases Kl/tot
=Kvo (Table 2, column 7), which is defined by equation (16),
K I / O = [ M ( PA)cI / O / ]  M ( PA)op 
(16)
and %M(PA)cl/tot= %M(PME-R)cl/o (Table2,column8). Similarly simple is the

situation with dPMEA2- because in this case an additional metal ion interaction,
2−
next to the one with the -PO 3 -group, must occur with the adenine residue and
it was previously concluded [11]that this is the N7 site; hence, here equilibrium
(2) applies. Consequently, for the M(dPMEA) complexes it holds Kl/tot = Kl/N7, as
defined by equation (17),
K l / N 7 = [ M ( PA)cl / N 7 ] /  M ( PA)op 
(17)
and %M(PA)cl/tot=%M(dPMEA)cl/N7 (Table 2, columns 7 and 8).

It is evident that the situation for the complexes formed with PMEA2- and
9,8aPMEA> is more complicated, since more possibilities for the formation of
closed isomers exist, and that these possibilities materialize at least in part is evi-
dent from the observed rather large stability increases, log ∆ M / PA (Table 2, column
6), and also from the high formation degrees calculated for %M(PA)cl/tot. Fur-
thermore, it is rcvealing to see that the values given in column 8 of Table 2 for
%M(PMEA)cvtot and %M(9,8aPMEA)cI/tot (entries and 2) are for a given metal ion
very similar or even identical within their error limits.
Formation Degrees of the Four Isomers Existing in

Equilibrium for the M(PMEA) and M(9,8aPMEA) Species
Up to now the Cu2+/PMEA system is the one most thoroughly studied. In-
deed, it had originally been proven [14] that three isomers are important for
the Cu(PMEA) system [7]: (/) An ’open’ isomer, Cu(PMEA)op, in which the
metal ion is solely coordinated to the phosphonate group; (ii) an isomer which
involves the ether oxygen (see Figure 1) as shown in equilibrium (1), designated
as Cu(PMEA)cl/o; and (iii) an isomer in which not only a 5-membered chelate but
in addition a 7-membered one involving N3 exists, i.e. Cu(PMEA)cvom3. More
recently [11] evidence was provided that there is a fourth isomer, a minority spe-
cies, inwhich the phosphonate-coordinated Cu2+ interacts with N7 of the adenine
residue forming a macrochelate, Cu(PMEA)dmT, as indicated in equilibrium (2).
In this context it is important to emphasize that for steric reasons no macrochelate
involving only N3 can be formed by PMEA2-and Cu2+ [2a]. If one tries to form
such a species with molecular models, one automatically forces the ether oxygen
into the coordination sphere of the metal ion, giving rise to the already men-
tioned Cu(PMEA)vom3 isomer [2a]. If one summarizes all these results then the
simple equilibrium (7a) must be replaced for the Cu(PMEA) system by the rather
complicated equilibrium scheme (3) already introduced in Section 1. Ofcourse,
exactly the same reasonings also apply to the PMEA2-complexes formed with Ni2+
and Zn2+ as well as for the M(9,8aPMEA) species. For these systems a quantitative
evaluation toward the formation degree of the various isomers needs now to be
carried out.
The four equilibrium constants seen in scheme (3) are defined by the already
mentioned equations (16) and (17) together with the also necessary equations
(18) and (19):
(
K MM ( PA)op =  M ( PA)op  /  M 2+   PA2−  (18) )
K I / O / N 3 = [ M ( PA)cI / O / NM 3 ] / [ M ( PA)cI / O ] (19)
With these definitions the measured overall stability constant (eq. (7b)) can be
redefined as given in equations (20a)-(20d):
K MM ( PA) =
[ M ( PA)] (20a)
 M 2+   PA2− 
 M ( PA)op  + [ M ( PA)cl / N 7 ] + [ M ( PA)cl / O ] + [ M ( PA)cl / O / N 3 ]

= (20b)
 M   PA 
2+ 2−
= K MM ( PA)op + K I / N 7 ⋅ K MM ( PA)op + K I / O ⋅ K MM ( PA)op + K I / O / N 3 ⋅ K I / O ⋅ K MM ( PA)op (20c)
= K MM ( PA)op (1 + K1/ N 7 + K I / O + K I / O ⋅ K I / O / N 3 )
(20d)
The connection between the overall intramolecular equilibrium constant Kl/

tot already introduced in Section 3.4, and the accessible stability enhancement
(eq. (12)) is given by equations (21a) -(21e):
K MM ( PA)
K l / tot = M
− 1 = 10log ∆M / PA − 1 (21a)
K M ( PA )op
[ M ( PA)cl /tot ]
=  (21b)
 M ( PA)op 
[ M ( PA)cl / N 7 ] + [ M ( PA)cl /O ] + [ M ( PA)cl /O / N 3 ]
= (21c)
 M ( PA)op 
= K I / N 7 + K I / O + K I / O / N 3 ⋅ K I / O (21d)
= K I / N 7 + K I / O (1 + K I / O / N 3 ) (21e)
Values for Kl/tot were already calculated with equations (12) and (14) in Sec-
tion 3.4; they are listed in column 7 of Table 2 (entries and 2). The relation
between Kl/tot and the other three intramolecular equilibrium constants follows
from equations (2 b) and (2 c). Based on the reasonable assumption [7] that
the stability of the M(PA)cl/o isomer, where Pa2-= PMEA2- or 9,8aPMEA2-,
is well represented by that of the 5-membered M(PME-R)cl/o species (Figure
1) and the stability of the M(PA)cl/N7 isomer by that of the M(dPMEA)cl/N7
macrochelate, values for Kl/o, which define the position of equilibrium (1), and
Kl/N7, which refer to equilibrium (2), are also known (see the second to the last
paragraph in Section 3.4). Hence, the only unknown constant in equation (21e)
is Ki/o/N3 (eq.(19)) and thus values for this constant can be obtained, and con-
sequently, the formation degrees for all four isomers appearing in scheme (3) can
now be calculated. The corresponding results are summarized in Table 3 for the
M(PMEA) and M(9,8aPMEA) systems; as far as the error limits are concerned it
needs to be emphasized that three times the standard errors (3σ) are given.
From Table 3 it is evident that Cu(PMEA) and Cu(9,8aPMEA) (entries b
and 2b) have practically identical properties: The Cu(PA) cI/O/N3 species with
the 5-and 7-membered chelate rings dominate with formation degrees of about
45% followed by Cu(PA)cI/O with about 30%. As far as Cu(PMEA) cI/O/N3
is concerned, the result with 41 ± 12% is within the error limit identical with
the previously obtained 49 ± 10% where the formation of the fourth isomer,
Cu(PMEA) cI/N7, had not been taken into account [5,7]. This demonstrates
immediately that the Cu(PA)cI/N7 isomer must be a minority species; indeed,
the present calculations show that the formation degrees of Cu(PMEA)cI/N7 and
Cu(9,8aPMEA)cI/N7 amount only to about 7% (see also ref. [11]).
It is interesting to see that for the Ni(PMEA) and Zn(PMEA) systems about
50% each exist as the open isomer and the remaining half of the species is present
as chelates (Table3, entries a and c). In the case of Ni(PMEA) all three chelated
isomers occur with comparable concentrations though the formation degrees
of Ni(PMEA)cI/o and Ni(PMEA)cl/O/N3 appear to be slightly favored. With
Zn(PMEA) the Zn(PMEA)cI/o isomer seems to be the dominating species, the
formation degrees of the other chelates being zero within the error limits; here it
should be recalled that the overall stability constant for Zn(PMEA) is an estimate
only (Table 2, entry e) [5].
For Zn(9,SaPMEA) (Table 3; entry 2c) the results are more clear-cut since
in this case the overall stability constant of the complex could actually be mea-
sured (see Section 2.3): Again the Zn(9,8aPMEA)cI/o chelate dominates. How-
ever, in this case it may be helpful to rewrite the results for Zn(9,8aPMEA)cI/O,
Zn(9,8aPMEA)cI/N7 and Zn(9,SaPMEA)cI/O/N3 with one standard deviation
(lσ) only, that is 32 ± 4, 10 ±7, and 24 ± 9%, respectively. This view confirms that
Zn(9,8aPMEA) cI/o dominates but that Zn(9,8aPMEA) cI/O/N7 most likely

also exists, whereas Zn(9,SaPMEA) cI/N7 is definitely also for this system a mi-
nority species. The great similarity between the Zn(PMEA) and Zn(9,8aPMEA)
systems is evident, despite all shortcomings, from a comparison of the values in
entries c and 2c of Table 3. This is also true for the Ni(PMEA) and Ni(9,SaPMEA)
systems for which the values seen in entries a and 2a of Table 3 overlap within
their error limits.
Conclusions
The presented results prove that systems in which four different isomers occur in
equilibrium in solution can be treated in a quantitative way. They prove further
that both N3 and N7 of an adenine residue may bind to metal ions provided
primary binding sites promoting a favorable steric orientation are available. With
regard to nucleic acids this result is of relevance; in fact, that the more basic N7
[27] is suited for such purposes is by now general knowledge [12,39] where as this
property of N3 has only been recognized more recently 14, 27b, 35, 36a, 41].
Furthermore, it is astonishing to see how similar the coordinating properties of
the two nucleotide analogues PMEA2- and 9,8aPMEA2- (Figure 1) are towards
Ni2+, Cu2+ and Zn2+. On the other hand, this observation complements the
fact that both acyclic-nucleoside phosphonate analogues exhibit antiviral activity
[1,16,17]. Therefore, it is interesting to note that the coordination chemistry of
8-[2(phosphonomethoxy)ethyl] adenine (8,SaPMEA2-) differs [42] from the one
described herein, and that indeed this nucleotide analogue does not show any
useful biological activity [16,17].
Acknowledgements
The competent technical assistance of Mrs. Rita Baumbusch and Mrs. Astrid Si-
gel in the preparation of this manuscript, the help of Dr. Larisa E. Kapinos with
the spectrophotometric experiments, and stimulating discussions with members
of the COST D20 programme are gratefully acknowledged. This study was sup-
ported by the Swiss National Science Foundation (H.S.) and the Programme of
Targeted Projects ($4055109) of the Academy of Sciences of the Czech Republic
(A.H.) as well as within the COST D20 programme by the Swiss Federal Office
for Education and Science (H.S.) and the Ministry of Education of the Czech
Republic (D.20.002; A.H.). This study also received support from the University
of Basel and it is further part of a research project (No. 4055905) of the Institute
of Organic Chemistry and Biochemistry (IOCB) in Prague.
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Inorganic Speciation of
Dissolved Elements in
Seawater: The Influence of Ph
on Concentration Ratios
Robert H. Byrne
Assessments of inorganic elemental speciation in seawater span the past four

decades. Experimentation, compilation and critical review of equilibrium
data over the past forty years have, in particular, considerably improved our
understanding of cation hydrolysis and the complexation of cations by car-
bonate ions in solution. Through experimental investigations and critical
evaluation it is now known that more than forty elements have seawater
speciation schemes that are strongly influenced by pH. In the present work,
the speciation of the elements in seawater is summarized in a manner that
highlights the significance of pH variations. For elements that have pH-de-
pendent species concentration ratios, this work summarizes equilibrium data
(S = 35, t = 25°C) that can be used to assess regions of dominance and
relative species concentrations. Concentration ratios of complex species are
expressed in the form log[A]/[B] = pH - C where brackets denote species con-

centrations in solution, A and B are species important at higher (A) and lower
(B) solution pH, and C is a constant dependent on salinity, temperature and
pressure. In the case of equilibria involving complex oxy-anions (MOx(OH)y)
or hydroxy complexes (M(OH)n), C is written as pKn = -log Kn or pKn* = -log
Kn* respectively, where Kn and Kn* are equilibrium constants. For equilibria
involving carbonate complexation, the constant C is written as pQ = -log(K
1 1
2 Kn [HCO3-]) where K 2 is the HCO3 - dissociation constant, Kn is a cat-
ion complexation constant and [HCO3-] is approximated as 1.9 × 10-3 mo-
lar. Equilibrium data expressed in this manner clearly show dominant species
transitions, ranges of dominance, and relative concentrations at any pH.
Introduction
Solution speciation exerts important controls on chemical behavior. Speciation is
known to influence solubility, membrane transport and bioavailability, adsorptive
phenomena and oceanic residence times, volatility, oxidation/reduction behavior,
and even physical properties of solutions such as sound attenuation. In recogni-
tion of such influences, substantial efforts have been made to characterize the
chemical speciation of elements in seawater. While assessments of organic spe-
ciation have dominantly been obtained using modern voltammetric procedures
and, as such, have a relatively short history, assessments of inorganic speciation
typically involve a wide variety of analytical procedures that have been employed
over many decades.
Assessments of the inorganic speciation of seawater began with attempts to
determine dominant chemical forms in seawater based on available thermody-
namic data. Early compilations of Principal Species dominantly involved (a)
simple hydrated cations and anions (e.g. Na+, Ca2+, Cl-, F-), (b) ion pairs with
sulfate (e.g. MgSO 04 and CaSO 04 ), (c) fully hydrolyzed elements (e.g. HmPO4
2− 2−
m-3, HmAsO4m-3, MoO4 and WO4 ) and (d) chloride complexes (e.g. AuCl2-,
2−
HgCl44 ). While it was noted [1,2] that hydroxide complexes were important for
all ions with oxidation numbers greater than two, hydroxide complexes were no-
tably absent in Principal Species tabulations until the following decade.
The thermodynamic data compilations of Sillén and Martell catalyzed rapid
advances in equilibrium models of seawater speciation. These works were followed
by additional compilations that were critically important to modern sea-water
speciation assessments. In view of these developments, and additional extensive
experimental analyses appropriate to seawater, Principal Species assessments ten
to fifteen years after the pioneering work of Sillén demonstrated a much improved
awareness of the importance of hydrolysis in elemental speciation.
Inorganic Speciation of Dissolved Elements in Seawater 207
An additional major speciation assessment provided a greatly improved, com-

prehensive view of inorganic complexation in seawater. Based on the analogous
characteristics of metal complexation by carbonate and oxalate, Turner et al. con-
cluded that rare earth element complexation in seawater is dominated by car-
bonate. Subsequently, as the result of approximately twenty years of progress in
seawater speciation, the Principal Species assessment of Bruland listed seventeen
elements with carbonate-dominated Principal Species.
Speciation Calculations
Based on currently available data, Principal Species for a substantial portion of
the periodic table (through atomic number 103) are thought to be controlled or
influenced by pH. The main objective of the present work is a review of Principal
Inorganic Species for the elements in seawater. The principal focus of this work is
an assessment of the influence of pH on inorganic speciation.
The Principal Species assessment in this work differs from previous presenta-
tion formats in its objective of providing a simple quantitative means of assessing
Principal Species variations with changes in pH. Stepwise equilibrium constants
provide a simple means of assessing species concentration ratios as a function of
pH. In the case of equilibria involving simple protonation of complex anions,
MOx(OH)yn-, stepwise equilibrium constants are expressed in the form
 H +   H m Am − n 
= K n − m (1)
 H m +1 Am +1− n 
where An- = MOx(OH)yn-. Consequently,
 H m Am − n 
log + log  H +  = log K n − m
 H m +1 A m +1− n

and
log  H m Am − n  /  H m +1 Am +1− n  = − pK n − m + pH
(2)
where pH = -log [H ] and pKn-m = -log Kn-m. In the case of simple stepwise hydro-
+
lysis equilibria,
M(OH)n-1 +H2O⇔M(OH)n + H+,
stepwise hydrolysis constants are written as
[ M (OH )n ]  H + 
K *
= (3)
n
[ M (OH )n−1 ]
whereupon,
log
[ M (OH )n ] = log K * − log  H + 
[ M (OH )n−1 ] n  
and
[ M (OH )n ] = − pK * + pH
log (4)
[ M (OH )n−1 ] n
In the case of equilibria involving carbonate, due to the near constancy of

HCO3- concentrations in seawater, equilibria can be conveniently written in the
following form
M(CO3)n-1+HCO3-⇔M(CO3)n + H+
and
[ M (CO3 )n ] = ( K  HCO − )( K / [ H + ]−1 )

n  3  (5)
[ M (CO3 )n−1 ] 2
where
 H +  CO32−  [ M (CO3 )n ]
K = and K n =
/
2
 HCO3 
−
[ M (CO3 )n−1 ] CO32− 
/
Using the dissociation constant of HCO3- in seawater (K 2 ), and Kn values
appropriate to various carbonate complexation equilibria in seawater, the relative
concentrations of M(CO3)n and M(CO3)n-1 can be written as
log[M(CO3)n]/[M(CO3)n-1] = -pQn+pH(6)
/
where log Q = log(KnK 2 [HCO3-]) = -pQn and [HCO3-] is assumed to be well
approximated as 1.9 × 10-3 M (i.e. log[HCO3-] = -2.72).
Based on equilibrium data compilations including Smith and Martell, Martell
and Smith, Baes and Mesmer, Turner et al., Byrne et al., and Liu and Byrne, Table
1 provides a compilation of pKn, pKn* and pQn data, and equilibrium speciation
schemes appropriate to seawater (S = 35) at 25°C. The first two columns of Table
1 provide each element’s atomic number and identity. The third column provides
either (a) each element’s dominant forms and speciation, or (b) the chemical spe-
cies whose relative concentrations are to be evaluated using the data in column
4. As an example of the use of Table 1, the entry for Be indicates that the con-
centrations of Be2+ and BeOH+ are equal in seawater (25°C) at pH 5.69 and
the concentrations of Be(OH)+ and Be(OH)20 are equal at pH 8.25. Hence,
at pH 8.0 BeOH+ is the dominant species and the ratios [Be2+]/[BeOH+] and
[Be(OH)20]/[BeOH+] are calculated as 10-2.31 and 10-0.25. The pK* and pQ
entries for CuII indicate that
log([CuOH+]/[Cu2+]) = -8.11 + pH
and
log([CuCO30]/[Cu2+]) = -6.93 + pH
As such, it is immediately seen that Cu2+ is a minor species relative to Cu-

CO30 in seawater (e.g. [CuCO30]/[Cu2+]  18.6 at pH 8.2) and that the Cu-
CO30/CuOH+ concentration ratio is approximately 15 (log 15  1.18 = 8.11
- 6.93), independent of pH.
Table 1. A compilation of pKn, pKn*, pQn data and equilibrium speciation schemes appropriate to seawater
(S 35) at 25°C. Equilibrium constants are expressed on the free hydrogen ion concentration scale.
Discussion
Table 1 indicates that the elements in group 1 (H, Li, Na, K, Cs, Rb) exist promi-
nently as free hydrated cations. About 1% or less of each metal is ion paired with
sulfate ([MSO4-]/[M+] ~0.01). Hydrogen ions are an exception to this generaliza-
tion. The HSO4-/H+ concentration ratio in seawater is approximately 0.3.
Group 2 elements (Be, Mg, Ca, Sr, Be) are more strongly ion paired with
SO42- than most of the group 1 ions. Mg2+ is approximately 10% ion paired
with SO42- and the extent of SO42- ion pairing increases somewhat for the
heavier members of the group. Be2+ is the only member of group 2 with an ionic
radius sufficiently small to induce extensive hydrolysis. The pK* values listed for
Be in Table 1 indicate that BeOH+ is the dominant form of beryllium except at
high pH. With a normal seawater pH range between approximately 7.4 and 8.35
(on the free hydrogen ion concentration scale) the Be(OH)+/Be2+ concentration
ratio is never smaller than fifty.
The pK* and pQ compilations in Table 1 demonstrate that all group 3 ele-
ments (Sc, Y and La through Lu) are strongly complexed in seawater. Sc is the
only group 3 metal that is strongly hydrolyzed. At pH 8.0 (i.e., 1.6 pH units above
the Sc pK3* and 1.6 units below pK4*) the dominant form of ScIII is Sc(OH)30
and the Sc(OH)30/Sc(OH)2+ and Sc(OH)30/Sc(OH)4-concentration ratios are
both equal to approximately 40. Yttrium and the rare earth elements (Y and La-
Lu) are dominantly complexed by carbonate. The pQn values for these elements
shown in Table 1, calculated using the results of Liu and Byrne, [15] indicate that
MCO3+ is generally the dominant form for the lighter elements while M(CO3)2-
is dominant for the heavier elements. LaCO3+ is the dominant form of La even
at the highest pH of seawater (pH  8.35, pQ2 = 8.47) and Lu(CO3)2- is the
dominant form of Lu if pH > pQ2 = 7.42.
Group 4 elements (Ti, Zr, Hf ) are all strongly hydrolyzed. Ti(OH)40 is the
dominant form of Ti over a wide range of pH (pH > 2.5). The speciation charac-
teristics of Zr and Hf are very similar. Zr(OH)5- is the dominant form of ZrIV
above pH 5.99 and Hf(OH)5- is the dominant form of HfIV above pH 6.19.
Thus, for both ZrIV and HfIV the uncharged species, M(OH)40 is a significant
but minor species. At the lowest pH of seawater the Zr(OH)40/Zr(OH)5- and

Hf(OH)40/Hf(OH)5-concentration ratios are approximately 0.04 and 0.06 re-
spectively.
Group 5 elements (V, Nb, Ta) are strongly hydrolyzed. With the smallest ionic
radius of these three elements, VV is very strongly hydrolyzed. VO3(OH)2- is
the dominant form of VV above pH 7.4. Since the pK3 value for VO3(OH)2-/
VO43- is nearly two units higher than that for HPO42-/PO43- at zero ionic
strength, only VO2(OH)2- and VO3(OH)2- appear to be relatively abundant
within the normal pH range of seawater. Nb(OH)6- is the dominant form of
NbV above pH 7.4, and since the Nb(OH)4+/Nb(OH)50 concentration ratio
in seawater is smaller than 10-8 only Nb(OH)50 and Nb(OH)6- are significant
species in seawater. Ta(OH)50 is the dominant form of TaV in seawater. The
Ta(OH)6-/Ta(OH)50 concentration ratio is only on the order of 0.06 at the
highest pH of seawater and, as is the case for Nb, cationic species are unimportant
(Ta(OH))4+/Ta(OH)50 < 10-8). Thus, dominant forms for the group 5 elements
are VO3(OH)2-, Nb(OH)6- and Ta(OH)50.
Group 6 elements (Cr, Mo, W) are all strongly hydrolyzed. Mo and W exist
solely in the VI state, while Cr is found in seawater with oxidation numbers VI
and III. Mo and W have dominant species transitions near pH 3.5 (pK2 = 3.5),
while the HCrO4-/CrCO42- transition for CrVI occurs near pH 5.7. As such,
in the VI state, all three elements dominantly exist as MO42- and ion pairs with
Mg2+, Ca2+, etc. CrIII is strongly hydrolyzed and, with a pK3* value near 8.3,
Cr(OH)2+ is more abundant than Cr(OH)30 except at high pH.
Group 7 elements (Mn, Tc, Re) have dissimilar group chemistries due to dif-
ferences in favored oxidation numbers. TcVII and ReVII exist in solution solely
as TcO4- and ReO4-and should be exceptionally unreactive. Mn has two favored
oxidation states with highly dissimilar characteristics. MnII exists in seawater
dominantly as free hydrated ions (Mn2+) and ion pairs with Cl-. In the IV oxida-
tion state Mn, as MnO2(s), is highly insoluble. Thus, oxidation state transforma-
tions strongly influence Mn behavior in seawater.
Elements in group 8 (Fe, Ru, Os) have diverse and generally poorly under-
stood seawater chemistries. Fe exists in seawater dominantly as FeIII and, to a
lesser extent, as FeII. Iron in the II oxidation number has a solution chemistry
very similar to those of other MII ions in period 4. MnII, FeII, CoII, NiII, and
ZnII are all weakly ion paired with Cl-and are present in seawater dominant-
ly as free hydrated ions. As FeIII, iron is strongly hydrolyzed. Table 1 indicates
that the dominant form of FeIII throughout the normal pH range of seawater is
Fe(OH)30. This conclusion is somewhat controversial because (a) iron biogeo-
chemistry is important and intensively investigated, and (b) only one somewhat
problematic analytical procedure (solubility analysis) has been extensively used to
investigate the Fe(OH)2+/Fe(OH)30 and Fe(OH)30/Fe(OH)4- transitions. The

characteristics of Ru and Os speciation in seawater are very poorly understood.
It is probable that both elements are very strongly hydrolyzed. Based on available
data, Principal Species for Ru and Os are tentatively assigned as Ru(OH)n4-n
and OsO40.
Elements in group 9 (Co, Rh, Ir) have generally complex chemistries and are,
perhaps, only slightly better understood than the group 8 elements. The dominant
oxidation number for Co in seawater is II. CoII exists predominantly as Co2+ and
ion pairs with Cl-. RhIII is strongly complexed by chloride and is also strongly
hydrolyzed. Investigations in 0.5 M NaCl (Miller and Byrne, in progress) indicate
that RhIII forms a complex array of mixed ligand complexes (RhCla(OH)b3-
(a+b)). These investigations are challenging due to slow ligand exchange kinetics.
IrIII forms strong chloride complexes and, as in the case of RhIII, has slow ligand
exchange rates. In analogy with RhIII the Principal Species of IrIII are tentatively
assigned as IrCla(OH)b3-(a+b)) Both Rh and Ir occur in the IV oxidation state
but the solution chemistries of RhIV and IrIV are very poorly understood.
Group 10 elements (Ni, Pd, Pt) principally occur with oxidation number II.
Ni in seawater dominantly exists as free hydrated Ni2+. The chemistries of PdII
and PtII are similar and contrast sharply with the speciation of NiII. Pd and Pt
exist dominantly as PdCl42- and PtCl42- in seawater [16] but are, nonetheless,
significantly hydrolyzed as PdCl3OH2- and PtCl3OH2- at high pH. The poten-
tial importance of PtIVspecies in seawater is poorly understood.
The solution chemistries of group 11 elements (Cu, Ag, Au) in oxidation state
I are similar. CuI, AgI and AuI are strongly complexed with Cl- and hydrolysis
is insignificant. While Ag solely exists as AgI, Cu occurs dominantly as CuII in
oxygenated seawater and oxidation number III may be important for Au. CuII
chemistry is dominated by carbonate complexation, while AuIII speciation in
seawater appears (tentatively) to be dominated by mixed-ligand chlorohydroxy
complexes.
The speciation of group 12 metals (Zn, Cd, Hg), all in the II oxidation state,
involves a progression from very weak to very strong complexation. ZnII oc-
curs in seawater principally as Zn2+ and ZnCl+, CdII is moderately complexed
(CdCl+, CdCl20 and CdCl3-) and HgII is complexed very strongly as HgCl42-
and HgCl3-.
Group 13 elements (B, Al, Ga, In, Tl) in oxidation state III are very strongly
hydrolyzed. The Principal Species of these elements are B(OH)30, A1(OH)4-,
Ga(OH)4-, In(OH)30 and, tentatively, T1(OH)30. The speciation of each of
these elements is significantly pH dependent. For B, Al, Ga and In, each metal is
partitioned between uncharged and anionic forms. In contrast, TlIII appears to
be partitioned between T1(OH)30 and either T1C14- or mixed chlorohydroxy

species. Tl is unique among the group 13 metals in having a significant, and per-
haps dominant, I oxidation state. In this form TlI occurs principally as the free
hydrated Tl+ ion.
Group 14 elements (C, Si, Ge, Sn, Pb) have diverse speciation characteris-
tics. C is partitioned dominantly between CO32- and HCO3-, while for both Si
and Ge, uncharged forms are dominant (Si(OH)40 and Ge(OH)40) with lesser
concentrations (≤ 15%) of SiO(OH)3- and GeO(OH)3-. The sparse data avail-
able for assessment of SnIV speciation indicate that Sn(OH)40 is dominant over
a wide range of pH. The speciation of Pb is apparently unique among seawater
constituents in that PbII is partitioned between chloride complexes and carbonate
complexes. [17] The latter are dominant above pH 7.85.
Group 15 elements (N, P, As, Sb, Bi) in oxidation states V and III are strongly
hydrolyzed in seawater, and oxidation number V is favored relative to III for all
group 15 elements except Bi. Bi is present in seawater dominantly as Bi(OH)30.
NVand NIII exist solely as unprotonated NO3- and NO2-. The NH4+/NH30
ratio in seawater is significantly pH dependent and is always larger than ~10.
The dominant forms of PV and AsV in seawater are HPO42- and HAsO42-.
While H2PO4-/HPO42- and H2AsO4-/HAsO42- ratios are similar in seawater,
the PO43-/HPO42- ratio is substantially larger than that for AsO43-/HAsO42-.
SbV in seawater is present dominantly as Sb(OH)6-. The speciation of SbIII is
similar to that of BiIII in that Sb(OH)30 is dominant over a wide range of pH.
The As(OH)30/As(OH)4- ratio in seawater is pH dependent and generally larger
than six.
Group 16 elements include O, S, Se, Te and Po. O2- and OH- are found in
association with elements in every group of the periodic table except 1, 2 and
18. Dissolved O2 is very important in seawater because of its strong influence
on the oxidation/reduction behavior of solutions. The dominant form of OH- in
seawater is MgOH+. S exists in oxygenated seawater as SO42- and ion pairs with
group 1 and group 2 elements, and is not significantly protonated except at very
low pH. Se exists in seawater as both SeVI and SeIV. In the higher oxidation state
Se exists as SeO42- with protonation characteristics very similar to SO42- (pK
~1). As SeIV, selenium is partitioned between HSeO3- and SeO32- with the
former dominant at low pH and the latter dominant at high seawater pH. Te also
exists in seawater in the VI and IV oxidation states. In the case of TeVI, since pK
 7.35 for the Te(OH)60/TeO(OH)5- transition, TeO(OH)5- is the predomi-
nant species. For TeIV, pK  8.85 for the TeO(OH)3-/TeO2(OH)22- partition
and TeO(OH)3- is thereby predominant. Little is known about Po equilibria in
solution.
The group 17 elements (F, Cl, Br, I and At) exist with -I and V oxidation
numbers and the -I state is predominant for the lighter elements. F- occurs in
seawater as an approximately equimolar mixture of F- and MgF+. Cl and Br occur
dominantly as unassociated Cl- and Br-. The predominant oxidation number of I
is V. IV occurs as IO3- and is, to a small extent, ion paired with Mg2+. I- is found
in seawater at substantially lower concentrations than IO3-. Little is known about
the solution chemistry of highly radioactive At.
Overview of Speciation in Seawater

The results shown in Table 1 indicate that only a relatively small number of el-
ements have major species that do not involve hydrolyzed forms or carbonate
complexation. Such elements typically have oxidation numbers I, II and -I and are
found in groups 1, 2, 11, 12 and 17, and in period 4 (groups 7–10). Only eight
to nine elements have speciation schemes that strongly involve chloride compl-
exation. Such elements are found in groups 9 (RhIII and IrIII), 10 (PdII and PtII),
11 (CuI, AgI, AuI), 12 (CdII, HgII) and 14 (Pb11). However, of these elements
both Rh and Ir are importantly influenced by hydrolysis, Pd11 and Pt11 are sig-
nificantly influenced by hydrolysis, and Pb11 is strongly influenced by carbonate
complexation. Of the very large number of hydrolyzed elemental forms in seawa-
ter, approximately 17 have speciation schemes that are strongly pH dependent.
These elements include Be11, ScIII (groups 2 and 3), VV, NbV and TaV (group 5),
CrIII (group 6), FeIII and RuIII (group 8), RhIII and IrIII (group 9), BIII, AlIII, In111,
TlIII (group 13), CIV, SiIV and GeIV (group 14), PV and AsV (group 15), SeIV and
TeIV (group 16). As such, no elements with oxidation numbers 1, 2 and 6 (other
than Be11) have hydrolyzed forms whose relative concentrations are strongly pH
dependent within the normal pH range of seawater. Approximately 17 elements
with atomic numbers less than 92 (Cu, Pb, Y, and the lanthanides) have specia-
tion schemes that strongly involve or are dominated by carbonate complexation.
With the inclusion of UVI and the 9 actinides with oxidation number III (Am-
Lr), it is seen that carbonate complexation is important for a large portion of the
periodic table. Altogether, including elements strongly complexed by carbonate
and seventeen or more elements with pH dependent, hydrolyzed major species,
it is seen that the seawater speciation of more than forty elements is strongly in-
fluenced by pH.
Elements having exceptionally poorly understood speciation schemes in sea-
water include Ru and Os (group 8), Rh and Ir (group 9), Pt (group 10) and Au
(group 11). Speciation of the latter four elements may be dominated by a complex
array of chlorohydroxy complexes and, perhaps, a variety of types of halides. It
should also be anticipated that, for metals forming strong covalently bonded species,
complexation by ligands containing reduced sulfur may dramatically change fu-

ture Principal Species assessments. This concern is particularly relevant to Rh, Ir,
Pd, Pt, Au, Hg, and T1.
Appreciation of the role of carbonate in seawater complexation has grown
steadily over the past forty years. Experimental difficulties have impeded the prog-
ress of investigations involving the complexation of strongly hydrolyzed metals by
carbonate ions. Using new technologies, however, future improvements in car-
bonate complexation assessments are probable and the perceived role of carbonate
in trace element complexation may significantly expand.
In view of the importance of pH dependent speciation schemes for a wide
variety of elements in seawater, it is important to note that substantial uncertain-
ties remain in the equilibrium characterizations presented in Table 1. In many
cases, estimated speciation schemes must be based on data obtained using a single
analytical technique. It is, furthermore, particularly problematic when speciation
characterizations of strongly hydrolyzed metals at high pH are based solely on
solubility analyses. Among other complicating factors, the experimental solutions
used in solubility analyses generally have much higher metal concentrations than
are observed in the open ocean. Consequently, solubility analyses are conducive
to the formation of a more complex set of hydrolyzed species (e.g., polymers and
colloids) than are generally found in the oceans. Deconvolution of the data gener-
ated in such analyses can be challenging.
Acknowledgements
Thanks are given to Drs. Johan Schijf and Xuewu Liu for assistance in the prepa-
ration of this manuscript. The author also gratefully acknowledges the construc-
tive criticism of two anonymous reviewers.
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A Novel Method to Detect
Unlabeled Inorganic
Nanoparticles and Submicron
Particles in Tissue by
Sedimentation Field-Flow
Fractionation
Cassandra E. Deering, Soheyl Tadjiki, Shoeleh Assemi,

Jan D. Miller, Garold S. Yost and John M. Veranth
Abstract
A novel methodology to detect unlabeled inorganic nanoparticles was exper-
imentally demonstrated using a mixture of nano-sized (70 nm) and submi-
cron (250 nm) silicon dioxide particles added to mammalian tissue. The size
and concentration of environmentally relevant inorganic particles in a tissue
sample can be determined by a procedure consisting of matrix digestion, par-

ticle recovery by centrifugation, size separation by sedimentation field-flow
fractionation (SdFFF), and detection by light scattering.
Background
Laboratory nanoparticles that have been labeled by fluorescence, radioactiv-
ity, or rare elements have provided important information regarding nano-
particle uptake and translocation, but most nanomaterials that are commer-
cially produced for industrial and consumer applications do not contain a
specific label.
Methods
Both nitric acid digestion and enzyme digestion were tested with liver and
lung tissue as well as with cultured cells. Tissue processing with a mixture of
protease enzymes is preferred because it is applicable to a wide range of par-
ticle compositions. Samples were visualized via fluorescence microscopy and
transmission electron microscopy to validate the SdFFF results. We describe in
detail the tissue preparation procedures and discuss method sensitivity com-
pared to reported levels of nanoparticles in vivo.
Conclusion
Tissue digestion and SdFFF complement existing techniques by precise-
ly identifying unlabeled metal oxide nanoparticles and unambiguously dis-
tinguishing nanoparticles (diameter<100 nm) from both soluble compounds
and from larger particles of the same nominal elemental composition. This is
an exciting capability that can facilitate epidemiological and toxicological re-
search on natural and manufactured nanomaterials.
Background
The toxicology of nano-sized (diameter < 100 nm) particles is a topic of cur-
rent interest because there have been rapid advances in the synthesis of novel
nanomaterials for research, consumer, and industrial applications. Recent reviews
have discussed nanoparticle health effects [1,2]. The growing evidence of adverse
health effects from exposure to incidentally produced ultrafine particles from
combustion and atmospheric processes motivates concern about manufactured
nanomaterials. There is epidemiological evidence for cardiovascular effects of am-
bient ultrafine particulate matter (PM) [3]. Indications that inhaled particles can
translocate to the other organs [4] suggest a link between nanoparticles and neu-
rodegenerative diseases [5] and other systemic pathologies. Monitoring human
exposure to engineered nanoparticles (from air, water, food, consumer products,
A Novel Method to Detect Unlabeled Inorganic Nanoparticles 219
and soil), determining the rate of particle uptake by humans and food chain or-
ganisms, and measuring the resulting nanoparticle concentrations in target organs
are major challenges for nanoparticle toxicology studies [6].
Most nanoparticle uptake and translocation research has quantified nanopar-
ticles in vivo using some type of unique particle label. For example, nanopar-
ticle laboratory studies have included radioactive particles [4], trace metals such
as gold and iridium [7], and fluorescent particles [8]. However, the population
exposures most relevant to health involve the emissions or deliberate release of
high-production-volume manufactured nanomaterials and exposures to inciden-
tal nanoparticles, such as soot. Combustion emissions and manufactured powders
such as fumed silica, ultrafine titanium dioxide (TiO2), and similar industrial
materials rarely have a unique and easily detected label.
Examples of current techniques for measuring unlabeled inorganic nanopar-
ticles in animal organs include using electron microscopy to show localization
of TiO2 particles to the lung of rats [9] and using elemental analysis to show
the presence of manganese particles in neural tissue [10]. However, measuring
changes in the concentration of unlabeled particles in tissue with these techniques
is difficult. Extracting quantative information from TEM images is inexact and
elemental analysis does not distinguish particles from soluble forms and provides
no information on particle size.
A promising method to measure size and concentration of unlabeled nanopar-
ticles is through separation by field-flow fractionation (FFF), which was first devel-
oped in the 1960s for separating macromolecules, colloids, and particles [11,12].
FFF has been used for the measurement of numerous properties of macromol-
ecules and colloidal particles, including particle mass, size, and density. Caldwell
et al. reported seminal work applying FFF to detect protein-based particles in eye
lens cataracts [13]. FFF has been used to characterize natural aquatic colloids [14-
16], and perform size separation of single-walled carbon nanotubes [17].
FFF is similar to chromatography methods in that materials are separated
by transport velocity, but in place of a retention media the separation is carried
out in a thin, open channel with bulk flow in the longitudinal direction and a
separation field (centrifugal force, electric field, thermal gradients, or cross-flow)
in the perpendicular direction. Particles are driven to the wall by the separation
field and average particle distance from the wall is determined by the competition
between the separation field and the size-dependent diffusion of particles against
the concentration gradient. Since the narrow channel has a parabolic flow pro-
file (laminar flow), the particles farthest from the wall are in the highest velocity
streamlines and therefore travel the fastest. Sedimentation FFF (SdFFF) uses cen-
trifugal force to generate the separation field. The minimum detectable particle
size depends on the particle density and the maximum centrifugal force of the
SdFFF instrument [18,19]. Giddings provides a full derivation of the governing

equation and a graph of minimum resolvable diameter versus GΔρ for a typical
instrument channel geometry [20]. For example, with the instrument used in
this study, silica particles with a density of 2.0–2.65 g/ml and as small as ~22 nm
can be separated using the instrument’s maximum centrifugal force. For denser
particles such as gold, applying the same field can separate particles as small as 10
nm. Multiple detection techniques can be used simultaneously with FFF, includ-
ing fluorescence, ultra-violet absorption, and light scattering.
In this study tissue lysis and gradient centrifugation, well-established method-
ologies for the density-dependent separation of subcellular fractions, were adapted
to isolate oxide particles from biological samples. Particle isolation was combined
with SdFFF to detect and quantify unlabeled inorganic nanoparticles.
Results
Preliminary experiments were conducted to calibrate the instrumentation used
in this study and to determine the amount of particles needed for reliable detec-
tion. We used dilutions prepared from purchased silicon dioxide (SiO2) standards
(Postnova Analytics) with a known particle size, 70 nm, and a starting concentra-
tion of 25 mg/ml of particles suspended in aqueous surfactant. With the available
light scattering detector, reliable quantification of the standard could be obtained
with as few as 7 × 1010 particles per injected sample, which is equivalent to 25
μg of particle mass. Based on these data, our subsequent experimental work used
tissue samples containing 1–2 mg of particles. Using particle aliquots greater than
40 times the limit of detection enabled robust quantification in the experiments
to develop nanoparticle recovery protocols.
To allow comparison of the SdFFF results to established techniques, we per-
formed fluorescence microscopy and TEM on particle-treated cell culture samples
treated with rhodamine-labeled SiO2 particles and prepared by enzyme digestion
for SdFFF analysis. Figure 1A shows the starting 70-nm rhodamine labeled par-
ticles in aqueous surfactant visualized using TEM. The TEM confirms that the
manufacturer’s size is correct. Figure 1B demonstrates the interaction of 70-nm
rhodamine labeled particles with Human Aortic Endothelial Cells (HAECs). This
image shows localization of the nanoparticles to the cells and formation of micron
sized aggregates which are visible by light microscopy. During the first step of our
SdFFF particle analysis procedure, the cells are collected, lysed and treated with
Proteinase K to digest proteins. Figure 1C shows a microscopy image of the cell
debris and aggregated fluorescent particles at this stage of the isolation process. A
sample after the final cleanup prior to FFF analysis was analyzed via fluorescence
microscopy, however the particles were well dispersed, and not visible, by light
microscopy (data not shown). Figure 1D shows a TEM image of these particles
after final cleanup and dried onto a grid. The particles are aggregated and coated
by residual organic material. SdFFF separation of the particles form soluble com-
ponents yields the monodispersed particles can be seen by TEM (data not shown)
[19]. These rhodamine-labeled SiO2 particles have the same manufacturer, and
nominal size and surface functionalization as the unlabeled SiO2 particles used
for the SdFFF experiments.
Figure 1. A. TEM image of the as-received 70-nm rhodamine labeled SiO2 particles. B. Fluorescence microscope
image of human aortic endothelial cells (HAECs) treated with 70-nm rhodamine labeled SiO2 particles (10
μg/cm2) for 24 hrs. 20× objective, 200× magnification. Red= SiO2 particles; Blue = DAPI stained nucleus.
C. Fluorescence image of cell lysate containing the 70-nm rhodamine labled particles. 20× objective, 200×
magnification. D. TEM image of a dried aliquot of the final sample after cleanup for SdFFF containing 70-nm
rhodamine labeled SiO2 particles in dried residual material.
Figure 2 shows a fractogram demonstrating that unlabeled 70-nm particles

could be recovered from acid-digested rat liver tissue, sized by SdFFF, and quanti-
fied with a light scattering detector. The elution time of the particles in the acid-
digested sample agreed with the 70-nm standard in surfactant. A “void peak” is
commonly seen at the start of an FFF separation [18]. Caldwell et al. describes
the contents of this peak as containing soluble components as well as suspended
particles small enough to remain uniformly distributed across the channel even in
the presence of the field [13]. Liver tissue was used because it is non-fibrous and
digestion with concentrated nitric acid resulted in complete digestion of the tissue
with the fewest processing steps. Since the nitric acid process is limited to acid-
insoluble particles we next developed a more gentle tissue processing protocol
utilizing protease enzymes. Lung tissue was used because it is the primary target
in particle inhalation studies. Tissue processing method development experiments
(data not shown) led to the protocol described in Methods below which involved
the use of specific enzymes to digest the extracellular matrix, inclusion of an aque-
ous surfactant in all processing steps, and sonication to redisperse the particles
after centrifugation.
Figure 2. SdFFF fractogram of 70-nm SiO2 particles recovered from acid-digested rat liver.
To demonstrate the capability to distinguish nano-sized (diameter < 100 nm)

and submicron particles alone and in lung tissue, mixtures of two different sizes
of SiO2 particles were added either to aqueous surfactant (reference sample) or
added to homogenized lung tissue which was processed by the tissue digestion
procedure. Figure 3A shows light scattering versus time from SdFFF analysis of
the reference sample normalized to the largest peak. This sample contains the
70- and 250-nm manufactured SiO2 particles at a 2:1 mass ratio in aqueous sur-
factant. Figure 3B shows the enzyme-digested rat lung tissue containing 70- and
250-nm manufactured SiO2 particles at the same concentration as in the refer-
ence sample (2:1 ratio). The inset graph is a magnified version of the circled area
showing that we were clearly able to detect 2 particle sizes from a tissue sample.
In both figure 3A and 3B graph shows the expected bimodal distribution of SiO2
particles. The difference in the relative sizes of the 70- and 250-nm peaks in the
reference samples is due to the size-dependent sensitivity of the light scattering
detector. Rayleigh scattering theory predicts that scattering intensity from a single
particle varies with the dp6 where dp is particle geometric diameter. However,
the number of particles for a given mass increases inversely with the dp3, and the
mass ratio of the 70 to 250 nm particles was 2:1. Thus the expected ratio of peak
areas would be about 23:1. A similar experiment using a mixture of 80-nm and
500-nm particles in enzyme-digested rat lung tissue also produced the expected
bimodal fractogram (data not shown). The geometrical size of the 70-nm particles
was confirmed by transmission electron microscopy (TEM) of a sample collected
after the SdFFF separation [19].
Figure 3. A. SdFFF fractogram of the reference sample of 70 and 250-nm particles mixed in surfactant. The
secondary x-axis depicts the theoretical particle size corresponding to the elution time for two particle densities.
B. SdFFF fractogram of 70 and 250 nm particles isolated from homogenized lung tissue. The inset graph is the
circled area enlarged, emphasizing the 70 nm particle peak.
Well established FFF theory [11] allows the particle size corresponding to
a given elution time to be calculated directly from first principles [19]. For Sd-
FFF the calculation is based on measurable physical parameters of the apparatus,
the carrier fluid, and the particle density, and involves the equations for settling
velocity, particle diffusion rate, and laminar flow profile. Figure 3A shows the
theoretical particle sizes (top x-axis labels) corresponding to the measured elution
time (bottom x-axis labels) for two different particle densities. These assumed
densities, 2.65 and 2.0 g/cm3, correspond to quartz and the density of the 70-
nm particles obtained from the vendor datasheet. These assumed densities span a
reasonable range for various amorphous and crystalline forms of SiO2. As can be
seen from the differences between the two sets of theoretical sizes, the particle size
corresponding to a given elution time is not strongly dependent on the assumed
density. Thus nanoparticles can be distinguished from micron-sized particles even
when the particle composition and density are uncertain. For example, detec-
tion of a particle mode within the time range corresponding to SdFFF separation
of nano-sized particles for a plausible range of densities would provide useful
hypothesis-generating information in a toxicology study of environmental expo-
sures.
Particle recovery for the experiment in figure 3A and 3B can be estimated
from the integrated area under the curve for the SdFFF analysis of the tissue
sample and the reference sample [19]. Particle recovery in the enzyme digestion
processing was 30% for the 250 nm particles and 22% for the 70 nm particles.
These recoveries are representative of one set of experimental data.
Discussion
The goal of this study was to develop a technique that provides detailed informa-
tion on the size distribution of unlabeled submicron and nano-sized inorganic
particles in toxicology samples. Specifically, we wanted to be able show directly by
instrumental analysis whether visible particle clusters, such as are shown in Fig-
ure 1B, contain nano-sized primary particles. Elemental analysis and radioactive
labeled particles provide mass concentration data but not size data. Microscopy-
based techniques provide size data only after image analysis of a sufficient sample
to get accurate statistics. Manual image analysis is labor intensive and automated
image analysis is subject to artifacts from overlapping particles or poor contrast
from the background. SdFFF complements the available techniques by providing
detailed size distributions from each sample run.
The sample handling methods used this study involved particle dispersion by
ultrasound in the presence of a surfactant to demonstrate the presence of a mode
corresponding to the primary particle size of the test particle. Characterizing the
size distribution of particle aggregates in biological samples is a complex problem

that is outside the scope of this pilot study. Particle aggregation is a dynamic pro-
cess since aggregates are held together by weak surface forces. Aggregate size can
be changed by mechanical force as well as by changes in pH, ionic strength, and
concentration of surfactants.
A wide range of unlabeled submicron and nano-sized particles of inorganic
materials can potentially be detected in tissue samples by the methodology de-
scribed in this paper. All these proof-of-concept experiments used SiO2 particles,
but the technique is directly applicable to a much wider range of particle types,
size and shape. The requirements for particle detection by our technique are that
the particle be sufficiently dense compared to the carrier fluid, have a different
index of refraction from the carrier fluid, and be resistant to the reagents used for
tissue digestion and sample cleanup. These requirements are met by essentially all
inorganic oxides, pure metal, and elemental carbon-based particle types. Non-
spherical particles can also be separated using SdFFF, but mathematical prediction
of retention time is more complicated than for spheres. A study using rod-shaped
aggregates of latex particles showed that the SdFFF separation time is determined
by the maximum dimension of the particle rather than by any average size [21].
Thus, this robust methodology is suitable for use in a wide range of particle toxi-
cology studies that involve correlating biological effects with the concentration of
nanoparticles in target organs. The particle size distribution information furnished
by SdFFF separation will be uniquely applicable to comparisons of the biological
effects of solid particles versus the effects of soluble species, a question that cannot
be answered by elemental analysis of ashed or acid-digested samples.
The important question of quantifying human lung burden of combustion-
generated nanoparticles provides an example of how the sample preparation and
SdFFF separation techniques presented in this paper can be used to complement
other methods. Vehicle tailpipe emissions contain submicron particles of carbo-
naceous material from incomplete combustion and metal oxides from fuel and lu-
bricant additives and engine wear. The carbonaceous primary particulate includes
both low-volatility compounds, referred to as organic carbon, and essentially
non-volatile large polycyclic molecules, referred to as elemental carbon or black
carbon. Grigg et al. conducted a study that used light microscopy to measure
black material in lung macrophages of healthy children and correlated this lung
burden with lung function and modeled levels of particulate matter [22]. Light
microscopy measures the two-dimensional projected area of particle aggregates.
This approach is labor intensive, introduces artifacts from the image analysis, and
provides no information on the primary particle size distribution or the compo-
sition of the opaque material. Saxena et al. recently published a technique for
quantitative estimation of diesel and carbon black particles in lung cells based
on adapting the thermal-optical-transmittance analytical technique developed for

measuring organic and elemental carbon in air pollution samples [23]. This tech-
nique provides quantification of the low-volatility and non-volatile carbon by a
precise instrumental analysis method, but again provides no information on the
primary particle size distribution or on the metal oxide components. In contrast,
SdFFF analysis of tissue provides quantitative information on the particle size
distribution after dispersal of the recovered particles by sonication in surfactant.
Our tissue processing method has the potential of offering high sensitivity since
the centrifugation steps allow concentrating the particles from large volume of
digested tissue into a small aliquot for analysis. Sequential collection of samples
during a SdFFF run is a well established technique [18]. The collected samples,
which represent concentrated and size-segregated fractions of the initial particles,
can be further analyzed, for example by transmission electron microscopy or el-
emental analysis. Compared to the other approaches cited above, the tissue diges-
tion and SdFFF approach presented here provides the ability to analyze particle
size distribution in large samples, such as a whole lung, and provides information
on both carbonaceous and metal oxide particles. Carbonaceous combustion par-
ticles have lower density than the silicon dioxide used in this study, but analysis of
carbon black by SdFFF has been demonstrated [24,25]. With additional method
development this technique can become a useful tool for studying environmental
particle burdens in lungs. Little is known about the background level of naturally
formed nanoparticles, and this technique can also be applied to ecosystem studies
of nanoparticles in sentinel and food chain organisms.
Considerable future research will be needed to fully realize the potential of our
technique for nanoparticle characterization in toxicology studies. Specifically, im-
provements are needed to reduce particle losses during the enzyme digestion and
particle recovery steps of the tissue processing, to make the method sufficiently
reproducible, and to permit precise quantification of the nanoparticle burden per
weight of original tissue. This paper describes experiments done with relatively
high concentrations of the particles because our goal was to demonstrate proof-
of-concept. The limit of detection for 70 nm SiO2 particles was 25 μg of particles
per SdFFF analysis sample using a light scattering detector. This particle concen-
tration in tissue is within the range reported by in vitro and in vivo nanoparticle
toxicity studies. For example, a study of fine and nanoscale quartz particles report-
ed statistically significant responses with an intratracheal instillation dose of 1 mg/
kg which equated to an initial burden of about 140 μg of particles per lung [26].
In an inhalation study exposing mice, rats, and hamsters to ultrafine TiO2, the
particle burden in the lung was 1.6 mg TiO2/g of tissue immediately post expo-
sure [27]. Thus, the currently demonstrated enzyme digestion and FFF detection
methodology is applicable to nanoparticle toxicology studies that use superphysi-

ological doses. However, improvement in the method sensitivity will be needed
for use in toxicology studies that use environmentally relevant particle exposures.
The tissue processing protocol involves particle recovery by centrifugation and the
centrifugation process intrinsically allows a sample to be concentrated. Processing
larger initial tissue samples and recovering the particles in a small final volume is a
straightforward way to achieve detection of low particle concentrations in tissue.
Conclusion
The capability to detect nanoparticles and to distinguish particle size distribu-
tion for unlabeled SiO2 in a sample of mammalian lung tissue has been demon-
strated. We have shown that not only can we detect unlabeled SiO2 nanoparticles
isolated from rat lung and liver tissue, but more importantly, we distinguished
between nano- and submicron-sized particles isolated from the same tissue. The
combination of enzyme digestion of tissue with particle sizing by SdFFF is a novel
approach that will greatly facilitate measurements of natural and anthropogenic
nanoparticles in laboratory toxicology studies, ecological systems, and human
populations. This work introduces a new method to characterize the size distri-
bution of unlabeled inorganic particles in tissue which will be useful for studies
focused on the neurological and cardiovascular effects of environmental and oc-
cupational exposures to an important class of engineered nanomaterials.
Methods
Tissue Preparation
Figure 4 outlines the tissue preparation, particle addition and isolation, and sam-
ple cleanup. Briefly, whole lungs and livers were collected post-mortem from male
Sprague-Dawley rats in accordance with an IACUC-approved protocol and snap
frozen in liquid nitrogen. The tissue was then ground with a mortar and pestle
in liquid nitrogen. The powdered tissue was suspended in 3 ml per gram of tis-
sue of a low-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2,
0.02 M KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5
mM dithiolthreitol). The tissue was further processed by homogenization using a
PRO200 series portable homogenizer (ISC BioExpress, Kaysville, UT) at 30,000
rpm until there were no visible chunks and then transferred to a motor-driven
Teflon-glass homogenizer (Potter-Elvehjem), (Fisher Scientific) and run at 900
rpm for 2 full passes to ensure the tissue was thoroughly homogenized.
Figure 4. Schematic of the tissue sample preparation protocol.
Nanoparticle addition
We added aliquots of particle suspensions (typically 1–2.5 mg) to homogenized
lung or liver tissue. The addition of particles to homogenized tissue demonstrated
nanoparticle detection in a complex mixture of biological material without the
complications related to in vivo particle distribution and uptake and elimination.
Particles were 70 nm diameter SiO2 (Z-PS-SIL-004-0,07, Postnova Analytics
Landsberg, Germany) and 250 nm SiO2 (Alfa Aesar, Ward Hill, MA).
Enzyme Digestion
Collagenase (150 U/ml) and hyaluronidase (100 U/ml) (Sigma Aldrich) were
added to the homogenized tissue to break up the extracellular matrix. The mixture
was incubated overnight at 37°C with shaking. The particle-spiked tissue was then
sonicated using an Ultrasonic Processor (Cole-Parmer, Vernon Hills, Illinois) for
20 seconds (2 sec bursts). To further break down the proteins we incubated the
tissue with 200 μg/ml Proteinase K (Sigma Aldrich) in 0.5% SDS for 2 hrs at
65°C.
Particle Isolation
The tissue samples were then layered over a saturated sucrose cushion and centri-
fuged at 21,000 × g for 20 min in a micorcentrifuge (Eppendorf North America,
Westbury, NY). The pellets were resuspended in 0.1% FL-70 (Fischer Scientific)
and sonicated for 20 sec (2 sec bursts). Phenol was added in a 1:1 (v/v) ratio and
incubated with shaking for 5 min followed by another round of centrifugation.
The pellet was then resuspended and washed with 70% ethanol and centrifuged.
The final pellet was resuspended and sonicated in 0.1% FL-70 with 0.01% so-
dium azide (Sigma Aldrich).
SdFFF
Analysis of the final samples was done using a Postnova S101 particle fractionator
(Postnova Analytics, Salt Lake City, UT). The injected sample volume was 100 μl
using 0.1% FL-70 as the carrier fluid at a rate of 2 ml/min. The initial speed of
centrifugation was 1800 rpm and the final speed was 200 rpm. SdFFF run time
was typically 90 min. Detection was achieved with a light scattering detector
(Brookhaven, Holtsville, NY) at a 90° angle (690 nm laser). A companion paper
[19] provides further details of the SdFFF methodology.
Acid Digestion
Acid digestion was used as an alternative to the enzyme digestion protocol above.
The tissue sample was transferred to a glass test tube and an equal volume of 60%
nitric acid (Fisher Scientific) was added. The test tube was placed in a beaker of
hot (94°C) water for about 1 hour or until the tissue was completely digested. The
samples were then centrifuged and the pellet washed with dilute acid and finally
resuspended in 0.1% of the aqueous surfactant FL-70.
Cell Culture
Treatment of live cell cultures with particles was used as an alternative to adding
particles to homogenized tissue, described above. Human aortic endothelial cells
(HAEC, Cambrex, Bio Science Walkersville) were cultured in 5% CO2 at 37°C
in either a T-25 culture flask (Corning, Corning, NY) or a glass bottom culture
dish (MatTek Cultureware, Ashland, MA) in endothelial cell growth medium-2

(EGM-2, Cambrex, Bio Science Walkersville) until 90% confluent. Cells were
treated by replacing the media with 4 ml of fresh EGM-2 containing 25 μg/cm2
rhodamine-labeled 70 nm SiO2 (Z-PS-SIL-RFP-0,07 Postnova Analytics Lands-
berg, Germany) and incubated for 24 hrs. To harvest the cells and the attached
or internalized particles for experiments, the culture medium was removed and
the cells were washed with phosphate buffer saline (PBS). Following the removal
of PBS, 1 ml TrypLE enzyme (Invitrogen) was added and then removed after
one minute and incubated for 5 min. The cells were washed from the dish with
fresh media and collected by centrifugation at 200 g, resuspended in 500 μl 0.1%
FL-70 and sonicated with a probe for 20 seconds (2 sec bursts). The particles
and lysed cell contents were then visualized via fluorescence microscopy and then
processed via enzyme digestion starting with the Proteinase K step (Figure 4).
Fluorescence Microscopy
Cells were treated with rhodamine-labeled 70 nm SiO2 particles (Z-PS-SIL-RFP-
0,07; Postnova Analytics Landsberg, Germany) and fixed in ice cold 100% meth-
anol. The nuclei were stained with DAPI (Molecular Probes). The stained cells
were visualized using an Olympus 1 × 50 fluorescent microscope and a Hama-
matsu camera. Images were analyzed using ImageJ software.
Transmission Electron Microscopy (TEM)

Particle samples for TEM were prepared by washing the particles, concentrating
by centrifugation, and resuspending in high-purity water. A 5 μL aliquot was
placed on a formvar-coated copper grid and allowed to dry overnight. Samples
were imaged on a Philips Techni G2 electron microscope at 100 kV.
Competing Interests
Postnova is a manufacturer of SdFFF instrumentation. CED, JDM, GSY, JMV
have no competing financial interests.
Authors’ Contributions
CD, JV, GY developed the cell culture and tissue sample preparation methods.
SA, ST, and JM provided the Sd-FFF separation methods. All authors partici-
pated in the data analysis and manuscript preparation.
Acknowledgements
We would like the thank Postnova Analytics USA for technical assistance and
sample analysis, and Nancy Chandler at the Health Sciences Center Electron
Microscopy Core for the TEM imaging resources. Support was received from:
K25 ES011281, EPA-STAR 8317230 and a University of Utah Synergy Grant
for nanoparticle toxicology.
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Chemical Speciation of
Inorganic Compounds Under
Hydrothermal Conditions
M. M. Hoffmann, J. L. Fulton, J. G. Darab, E. A. Stern,

N. Sicron, B. D. Chapman and G. Seidler
XAFS
By employing XAFS spectroscopy we are seeking a better understanding of the
high-temperature aqueous chemistry and speciation of those components that
are troublesome to the treatment of the Hanford tank wastes: namely Cr, Re
(surrogate for Tc), Fe, Ru (surrogate for Pu), and NO3-/NO2-. After initial ex-
periments revealed that the high corrosivity of some of these aqueous systems
renders the original high-pressure/high-temperature XAFS flow cell unsuitable
of these systems, we have quickly implemented a new XAFS flow cell design
made from Pt/Ir alloy. Over the last 12 months we have been able to obtain
a number of groundbreaking results with this new XAFS cell, which we will
briefly highlight.
Chemical Speciation of Inorganic Compounds 235
Redox Chemistry
Figure 1 shows XANES spectra obtained at 400oC from 0.02 molal aqueous
Cr(NO3)3 solutions with and without 0.06 molal NO2-. The dominant peak in
the spectra is a signature for Cr(VI) as demonstrate by the additional spectrum
of solid Na2CrO4 4H2O. Hence, at 400oC the aqueous chrome is fully oxidized
from Cr(III) to Cr(VI) by NO3-/NO2-but is only to ~50% oxidized by NO3alone.
These spectra are to the best of our knowledge the first reported in situ spec-
troscopic observation of homogeneous aqueous redox chemistry at temperatures
beyond the critical temperature of water (>374oC). We also observed a time-de-
pendence for the growth of the Cr(VI) XANES peak and have therefore obtained
some kinetic information for this redox system as well.
In other studies we investigated the stability of aqueous ReO4- to high tem-
peratures (up to 400oC). The ReO4- was found to maintain the oxidation state
VII regardless of a wide range of solution pH. ReO4- remained stable to 400oC
even in the presence of the reducing agent NH4+.
We have also obtained information on the high-temperatures redox behavior
of aqueous Cu2+, another tank waste species that is prone to redox chemistry
during high temperature processing. We found aqueous solutions of CuBr2 to
be extremely corrosive. At high temperatures, Cu(II) has a strong tendency to be
reduced to Cu(I) by reaction with other metal species. With the new XAFS cell
we were able to obtain in situ results to 300oC for aqueous solutions of CuBr2
that show that the copper is present as Cu(I), most likely reduced by dissolving
Pt. High temperature spectra of Cu(NO3)2 and CuBr2 with added NH4Br were
also obtained.
Oligomerization Chemistry
Aqueous solutions of CrO42- are known to undergo oligomerization reactions
upon acidification. Isopolymetalates are also formed by the other row VI ele-
ments, Mo and W. These kind of polymerization processes are in general very
common in aqueous solutions as they apply for the precipitation mechanism of
hydroxides and oxides at basic pH conditions.
As the first benchmark experiments we investigated the isopolytungstate sys-
tem to high temperatures. The EXAFS spectra are very rich in information and
show large changes with both temperature and (starting) pH with a dramatic re-
duction in complexity between 200oC and 300oC. In order to better understand
and possibly quantify the observed spectral changes in the EXAFS we have turned
to complimentary IR spectroscopic investigations. These IR measurements re-
quire a very short optical path length thus a new IR cell was specifically designed
and built for this purpose. The combined results strongly indicate that besides the
tungstate monomer a second, yet unidentified, species of simple geometry must
be present at 300oC and starting pH value < 8. In contrast, recently acquired
XAFS spectra of aqueous chromate solutions to low pH values and high tem-
peratures show little change, indicating that the chromate remains tetrahedrally
coordinated with little or no changes in the Cr-O bond distances throughout all
investigated experimental conditions. We have also acquired XAFS spectra for
the molybdate systems to high temperatures enabling us to compare the high
temperature aqueous oligomerization chemistry between the row VI transition
elements.
Future Directions
Further work will continue the CrIII/CrVI -- NO3-/NO2- redox investigations.
These investigations will include a larger range of temperature conditions with at-
tempts to obtain more refined kinetic information. Our new IR cell will come in
handy to monitor the fate of the NO3-/NO2-anions and thereby obtain a complete
understanding of this redox system.
So far we have not studied aqueous iron solutions because these systems are
multi-phased and presently we are experimentally limited to systems that are ho-
mogeneous, i.e., single-phase. However, we plan to slightly modify the new IR
cell design allowing us to use this design as a suitable XAFS cell for multi-phased
systems including solid, liquid and gas. In particular, we hope to be able to also
monitor changes that occur 500oC in solid phases and map out phase equilibria
and oxidation state by x-ray imaging methods.
Recent improvements at the PNC-CAT beamline of the APS at the Argonne
National Laboratory have increased the range of x-ray beam energies to include
the high absorption edge Ru. Hence, we are now able to begin studies of aqueous
solutions of ruthenium.
Status
As highlighted above we have obtained a large set of new in situ results. The data
reduction and analysis is in various processing stages. However, our first results
on the tungstate and perrhenate systems have already been presented at the 8th
annual V. M. Goldschmidt conference, the EMSP conference and X-98 confer-
ence both last summer in Chicago, and in article form in Mineralogical Magazine
and Chemical Geology (to be published this fall). Our more recent findings will
be presented at the upcoming EMSP symposium at the ACS conference in New
Orleans as well as at the ICPWS conference in Toronto.
Chemical Speciation of Inorganic Compounds 237
As mentioned in our EMSP proposal we have also attempted to use quartz

capillaries as XAFS cell for out measurements. During these attempts we made
the unexpected observations that Ni2+ is absorbed by the capillary at 425oC,
most likely to form a nickel silicate. These findings prompted to envisage the use
of silica as a new selective sequestering method for aqueous ionic waste streams.
Figure 1. Comparison of high-temperature Cr solution spectra with solid Na2CrO4 ·4H2O. Time at 400oC was
approximately one hour.
Anomalous Diffuse Elastic X-ray Scattering

This effort has concentrated on building and commissioning the Rowland Circle
(RS) x-ray spectrometer for recording the elastic portion of the diffuse scattered
x-rays from the water solutions. These measurements will complement the XAFS
measurements discussed in Sect. A by adding the pair distribution function of
atoms about the ion of interest to larger distances.
During the last year the apparatus has been constructed and tested success-
fully. Presently the apparatus is being commissioned to optimize its operation.
The tests have shown that the reflected efficiency of the x-rays is about an order
of magnitude below the optimum and steps are being made to improve this ef-
ficiency. These include slightly damaging the bent silicon single crystal used for
focusing to increase its rocking curve width, and minimizing the variations in the
thickness of this same crystal to improve its focusing power. Even in its present
non-optimum state the RS is still useful for measuring the data required to obtain
the radial distribution function about dissolved ions in water.
Anomalous scattering measurements have been performed on Sr ions formed

by dissolving strontium bromide in water under ambient conditions. These mea-
surements have been made for the same cell configuration to be used under super-
critical conditions. This cell consists of a hollow cylinder of boron carbide (BC)
that is sealed to titanium tubing on both ends by a pressure fit. The Ti tubing
is then connected to the pressure pump that causes the supercritical solution to
flow through the cell. A heater is placed around the cell to produce the required
temperature. The anomalous scattering measurements have indicated the need to
make further improvements in monitoring and controlling the alignment of the
apparatus to minimize spurious variations. Scattering measurements have also
been made on pure water in the cell to investigate the bacgound signal from the cell.
Figure 1 shows the scattering data obtained for pure water in the cell. Note
that besides the diffuse scattering from the solution, sharp Bragg peaks coming
from the polycrystalline BC cell are also present. Efforts are being made to mini-
mize these sharp peaks, though, because they are so much sharper than the diffuse
scattering spectra, it is possible to remove this background reasonably reliably
from the fluid signal. The measured signal that is produced by scattering from
both the water and the cell is shown by the solid plus dotted plots. The scattering
from pure water is shown by the solid line where the sharp Bragg peaks have been
subtracted
Figure 2. Diffuse elastic scattering from pure water placed in the sample cell to be used for supercritical water
solution measurements. Dotted curve, Bragg peaks from cell. Solid curve, the contribution from the water
obtained by subtracting the cell contribution.
As implied above, the complete setup for measuring diffuse elastic x-ray scat-
tering from supercritical solutions has been assembled. The cells and their seals
have been successfully tested up to the maximum pressure. What remains for the
sample containing portion of the apparatus is to test it under supercritical condi-
tions by adding the elevated temperature.
Measurements of Particle
Masses of Inorganic Salt
Particles for Calibration of
Cloud Condensation Nuclei
Counters
M. Kuwata and Y. Kondo
Abstract
We measured the mobility equivalent critical dry diameter for cloud conden-
sation nuclei (CCN) activation (dc_me) and the particle mass of size-selected
(NH4)2SO4 and NaCl particles to calibrate a CCN counter (CCNC) pre-
cisely. The CCNC was operated downstream of a differential mobility ana-
lyzer (DMA) for the measurement of dc_me. The particle mass was measured
using an aerosol particle mass analyzer (APM) operated downstream of the
DMA. The measurement of particle mass was conducted for 50–150-nm
particles. Effective densities (ρeff) of (NH4)2SO4 particles were 1.67–1.75

g cm−3, which correspond to dynamic shape factors (χ) of 1.01–1.04. This
shows that (NH4)2SO4 particles are not completely spherical. In the case of
NaCl particles, ρeff was 1.75–1.99 g cm−3 and χ was 1.05–1.14, demon-
strating that the particle shape was non-spherical. Using these experimental
data, the volume equivalent critical dry diameter (dc_ve) was calculated, and
it was used as an input parameter for calculations of critical supersaturation
(S). Several thermodynamics models were used for the calculation of water ac-
tivity. When the Pitzer model was employed for the calculations, the critical
S calculated for (NH4)2SO4 and NaCl agreed to well within the uncertainty
of 2% (relative). This result demonstrates that the use of the Pitzer model for
the calibration of CCNCs gives the most accurate value of S.
Introduction
The number concentration of cloud condensation nuclei (CCN) is an important
parameter for cloud microphysics. The number concentration and size distribu-
tion of cloud droplets are affected by changes in the CCN number concentration.
Consequently, CCN number concentration is indirectly related to radiative forc-
ing and the hydrological cycle. Thus, it is important to measure the CCN num-
ber concentration and CCN activity of atmospheric particles precisely (Twomey,
1974; Lohmann and Feichter, 2005, and references therein).
CCN number concentration is measured using a CCN counter (CCNC).
Several types of CCNCs have been developed (McMurry, 2000; Nenes et al.,
2001; Roberts and Nenes, 2005). In most CCNCs, supersaturated conditions
are produced by creating a temperature difference on wetted walls. CCN-active
particles grow to large droplets in the artificial supersaturated environment. The
number of droplets is then counted using an optical particle counter (OPC) (e.g.,
Stratmann et al., 2004; Roberts and Nenes, 2005) or a charge coupled device
(CCD) camera (Otto et al., 2002). The most important parameter in CCN mea-
surement is the precise value of the supersaturation (S) inside the instrument,
which ensures compatibility with other studies (Seinfeld and Pan-dis, 2006).
S inside a CCNC can be calculated theoretically (e.g., Nenes et al., 2001;
Stratmann et al., 2004; Roberts and Nenes, 2005). These theoretical studies
are indispensable for developing CCNCs. However, theoretical results are not
sufficient for practical observations and experiments as the ideal instrument does
not exist and instrumental conditions can change with time. Therefore, routine
calibrations are required for the operation of CCNCs (e.g., Rose et al., 2008).
Unfortunately, there are no instruments that can measure S directly; thus, we have
Measurements of Particle Masses of Inorganic Salt Particles 241
to choose an alternative method for calibration. In most CCN studies, the critical
dry diameters (the threshold diameters for CCN activation, dc) of laboratory-
generated particles are measured using a CCNC connected to a differential mo-
bility analyzer (DMA) in tandem to calibrate the instruments. Then, the critical S
values corresponding to the observed dc values are calculated using Köhler theory.
(NH4)2SO4 is the most frequently used compound for calibration (e.g., Kumar
et al., 2003; VanReken et al., 2005), and some studies also use NaCl particles
(Rissman et al., 2007; Shilling et al., 2007; Rose et al., 2008). However, the criti-
cal S of (NH4)2SO4 particles is strongly dependent on thermodynamics models
(Kreidenweis et al., 2005), and the magnitude of the variation is large enough to
change the interpretation of the observation results (e.g., Mochida et al., 2006).
In the case of NaCl, the differences between thermodynamics models are not as
significant as those of (NH4)2SO4, and there is an excellent thermodynamics
model that is based on the various experimental data (Archer, 1992). However, it
is difficult to estimate the critical S of a DMA-selected NaCl particle because of
its non-spherical shape (Kelly and McMurry, 1992; Zelenyuk et al., 2006a). In
previous studies, S values calculated using the two different compounds did not
always agree, and the magnitude of the difference was up to 10% (relative) (e.g.,
Shilling et al., 2007). These discrepancies were possibly caused by the uncertain-
ties described above. Rose et al. (2008) have suggested that the shape factor of
NaCl particles varies between 1.0 and 1.08 based on measurements of the CCN
activity of NaCl particles. We have measured this quantity more accurately using
a more direct method.
In this study, we measured the masses of (NH4)2SO4 and NaCl particles
generated for the calibration of a CCNC using an aerosol particle mass analyzer
(APM). The APM can select particles according to their mass by virtue of the bal-
ance between electrostatic and centrifugal forces (Ehara et al., 1996). Thus, the
combination of a DMA and an APM (DMA-APM system) enables us to measure
the mass of DMA-selected particles, and we can obtain parameters related to
particle morphology such as χ (e.g., Park et al., 2004). Then, S values inside the
CCNC were calculated using the measured dc, particle mass, and several thermo-
dynamics models. The calculated S values are compared to investigate the consis-
tency of the experimental results of (NH4)2SO4 and NaCl particles.
Theoretical Background
The Relationship of dme and dve
In this section, we summarize the relationship between mobility diameter (dme)
and volume equivalent diameter (dve), because the conversion of dme to dve is
needed to prepare an input parameter for calculations based on Köhler theory

(Sect. 2.2). For spherical particles, dme is equal to dve. However, particles are not
always spherical. Using the effective density (ρeff), dme is related with dve by the
following equation (DeCarlo et al., 2004):
p p
mp = rm d ve3 = reff d me
3
(1)
6 6
or
ρeff
d ve = d
ρ m me (2)
3
where mp is particle mass, and ρm is the material density. In the DMA-APM sys-
tem, dme is known as it is prescribed by the DMA, and mp can be measured using
the APM. Thus, if we apply this experimental system to particles composed
 ρ 
of single compounds, we can obtain the conversion factor  3 eff  easily, as ρm
is a known parameter.  ρm 
 
dve and dme can also be related by χ, which is defined as the ratio of the drag force
acting on spherical particles with diameters of dve and dme. Under the equilib-
rium sphere approximation (ESA) (Dahneke, 1973), χ is defined by the following
equation (Kasper, 1982):
Z p (d ve ) C c (d ve ) d me
c= =
Z p (d me ) d ve C c (d me ) (3)
where Zp is the electrical mobility, and the slip correction factor (Cc) can be calcu-
lated from the following equation (Allen and Raabe, 1985):
æ æ öö
çç çç -0.009 ÷÷÷÷
2l ç ÷÷
C c = 1 + çç1.142 + 0.558exp ççç 2l ÷÷÷÷÷÷ (4)
d p çç çç d p ÷÷÷÷÷÷÷
èç èç øø
where λ is mean free path of air. Although ESA is a good approximation for slight-
ly non-spherical particles and it is useful to calculate χ from experimental results,
it is not always appropriate to use ESA for studies of χ in the transition regime
(χt ), especially for particles that are highly non-spherical (Dahneke, 1973).
For this purpose, the adjusted sphere approximation (ASA) was introduced
(Dahneke, 1973). Using ASA, χt can be written as follows (De-Carlo et al.,
2004):
Cc (d ve )
ct = cc
æcf ö
C c çç d ve ÷÷÷ (5)
èç cc ø÷
where, χf and χc denote χ in the free molecular regime and continuous regime,
respectively.
Köhler Theory
The water activity of an aqueous solution (aw) is equal to the saturation ratio of
water vapor (Robinson and Stokes, 2002)
Pw
= aw (6)
Pw0
where pw is the vapor pressure of water and p 0w is the saturation vapor pressure
of water. For an aqueous solution in gas-liquid equilibrium, pw is equal to the
equilibrium water vapor pressure of a solution having a flat surface. In the case of
particles, the equilibrium vapor pressure of the solution (pw_aerosol) is affected by the
curvature of the droplet (Kelvin effect). The magnitude of this effect is described
as follows (Seinfeld and Pandis, 2006)
Pp _ aerosol æ 4s M ÷ö
= exp ççç w ÷
÷
Pw çè RT rw D p ÷÷ø
(7)
where σ is surface tension, Mw is the molecular weight of water, R is the gas con-
stant, T is temperature, ρw is the density of water, and Dp is the droplet diameter.
Substituting Eq. (7) into (6), we get
æ ö
s=
P w _ aerosol
= a exp çç 4s M w ÷÷
w ÷
çç RT r D ÷÷
Pw0 è w pø
(8)
where s is the saturation ratio of water vapor. Thus, aw is needed to calculate s.
There are several expressions for the aw of solution. One of the most commonly
used expressions is the equation defining the molal osmotic coefficient (φ) (Rob-
inson and Stokes, 2002);
ln aw = −υmMwϕ (9)
where υ is the stoichiometric number of solute ions and molecules, and m (molal-
ity) is defined as follows:
ms
m= (10)
M s mw
In Eq. (10), ms is the mass of solute, Ms is molecular weight of solute, and mw
is the mass of water in aqueous solution. The van’t Hoff factor (i) is also frequently
used to express aw. The value i is defined as follows:
nw
aw =
nw + ins (11)
where nw and ni are the numbers of moles of water (solvent) and solute, respec-
tively (Pruppacher and Klett, 1997). As an example of another expression, Tang
and Munkelwitz (1994) and Tang (1996) expressed aw as a polynomial equation
with respect to the concentration of the solution (weight percent). Thermody-
namics models employed for the present study are summarized in Appendix B.
Among the models summarized in Appendix B, we regard that of Archer (1992)
as the most reliable, as it is based on a number of experimental results, including
the concentration range that is important for CCN activation (Clegg, 2007). We
next derive Köhler theory using φ. Similar equations can easily be obtained for
other expressions of aw.
Using Eqs. (8), (9), and (10), we get
4s M w M m
ln s = - uj w s
RT rw D p M s mw (12)
In Eq. (12), we need to know ms for the calculation of s. It is equal to mp when
the particle is composed of a single component. Then, Eq. (12) can be rewritten
using dve (Eq. 1),
4s M w M w rm d ve3
ln s = - uj
RT rw D p M s rw (D p3 - d ve3 ) (13)
When we calculate s of a single particle as a function of Dp, it has a maximum
value. S corresponding to this value is called the critical S. If the particle is subject-
ed to an S greater than the critical S, the particle can grow into a cloud droplet.
Table 1. Ms , ρm, and ν of (NH4)2SO4 and NaCl.
Experiment
Particle Generation and Classification
The experimental setup used in this study is shown in Fig. 1. Aqueous solutions
(∼0.1 weight %) of (NH4)2SO4 and NaCl were prepared and introduced into an
atomizer (TSI model 3076). Chemical properties of these compounds are sum-
marized in Table 1. Synthetic compressed air supplied from a gas cylinder was
used for this atomizer. Particles were dried by passing them through two diffusion
dryers (TSI model 3062) connected in tandem. Silica gel used for the diffusion
dryers was regenerated before each run. Then, particles were charged with a 241Am
neutralizer, and their size was selected by a DMA (TSI model 3081). The sheath
and sample flow rates of the DMA were set at 3.0 lpm and 0.3 lpm, respectively.
The size selection of the DMA was checked by measuring size distributions of
the polystyrene latex (PSL) particles listed in Table 2. The peak diameters of the
size-distributions agreed with the diameters of the PSL to within the errors given
by the manufacturers. In this paper, we report diameters by the set values of the
DMA. The random error in diameter estimated from the PSL measurements was
less than 0.5%.
CCN Measurement
The CCN measurement part of Fig. 1 was used to determine the mobility equiva-
lent critical dry diameter (dc_ me). A CCNC (Droplet Measurement Technologies,
DMT) (Roberts and Nenes, 2005) was used to measure CCN number concen-
tration, and a condensation particle counter (CPC: TSI model 3022) was used
for CN measurement. The sample flow from the DMA was mixed with dry com-
pressed particle-free air (0.5 lpm) to keep the sample flow rate of the DMA at
0.3 lpm. Dilution air was produced from air in the laboratory using a pure air
generator (PAG 003, ECO Physics) and a high-efficiency particulate air (HEPA)
filter. The flow rate of the dilution air was controlled by a mass flow controller.
The sample flow and the sheath flow rates of the CCNC were set to 0.045 lpm
and 0.455 lpm, respectively. Two temperature gradient (∆T ) conditions of the
thermal gradient chamber inside the CCNC were used as shown in Table 3 so
that the measurement was performed at two S values. Solenoid pumps used for
water circulation in the CCNC were replaced by external peristaltic pumps for
flow stabilization.
Table 2. List of PSL particles used for the calibration of the DMA and APM. a. Density given by the manufacturers
as a reference. b. Particle mass was calculated using the diameter range and density.
Figure 1. Experimental setup used in this study.
The air circulation system for the OPC drying system was plugged to stabilize
the airflow in the chamber. The reproducibility of dcmemeasurement was tested
using (NH4)2SO4 particles. The random errors associated with dc_me measure-
ment were 0.1 and 0.3 nm for ∆T 1 and ∆T 2, respectively. The influence of this
error on the critical S was calculated to be negligibly small (less than 0.001%,
absolute). Although, we employed the CCNC manufactured by DMT, the pres-
ent result is applicable for other types of CCNCs, as they are calibrated similarly
(e.g., Snider et al., 2006; Frank et al., 2007).
DMA-APM System
An APM (APM 302, KANOMAX JAPAN, Inc.) was employed to measure mp
of particles prescribed by the DMA. The mp selected by the APM is described as
follows (Ehara et al., 1996),
neV APM
mp = (14)
w 2r 2 ln (r2 / r1 )
where n is the number of charges, e is the elementary charge, VAPM is the voltage
applied to the APM, ω is the rotation speed, and r, r1, and r2 are the center, inner,
and outer radii of the APM operating space, respectively. Particle number concen-
tration downstream of the APM was measured by a CPC (TSI model 3022).
Figure 2. Example mass distribution of PSL particles prescribed by the DMA.
To calibrate mp and ρeff measurement by the DMA-APM system, the masses

of DMA-selected PSL particles were measured. Figure 2 shows an example of a
mass distribution of DMA-selected PSL particles. The peak of the distribution,
which corresponds to mp, was obtained by fitting the distribution by a Gaussian
function. The measured values of mp agreed with the calculated values within
the errors associated with PSL particles (Table 2). Density calculated using Eqs.
(14) and (1) (in this case, we can assume dve =dme, as PSL particles have spheri-
cal shape) agreed with the values given by the manufacturers to within 5%, and
this difference was corrected for inorganic salt particles. Linear interpolation was
employed for the correction, as the difference was size-dependent. Relative contri-
butions of the DMA and APM to the difference were not quantified. The random
error associated with the ρeff measurement by the DMA-APM system was 1%,
estimated from PSL measurements.
Table 3. Temperatures of the thermal gradient chamber in the CCNC. T 1, T 2, and T 3 correspond to the
temperatures at the top, middle, and the bottom of the chamber, respectively.
The measurement of ρeff was performed four times. RUN1 and RUN2 were
performed prior to CCN measurement. RUN3 was performed soon after (within
5 h) the CCN measurement at ∆T 1, and we concentrated on the size range that
is important for CCN activation under these conditions. Likewise, RUN4 was
performed soon after the CCN measurement at ∆T 2.

dc_me of Inorganic Salt Particles
Figure 3 shows experimental results for the CCN measurement of (NH4)2SO4
and NaCl particles. CCN/CN ratios monotonically increase with increasing di-
ameter and approach unity. The CCN/CN ratios are fitted by a sigmoid function
(Eq. 15):
CCN b
=a+
CN æ d c _ me - d me ÷ö (15)
1 + exp çç ÷÷
çè c ø
where a, b, and c are constants determined by the fitting. dc_me values were 115.2
nm (∆T 1) and 75.3 nm (∆T 2) for (NH4)2SO4 and 94.7 nm (∆T 1) and 63.2
nm (∆T 2) for NaCl, respectively.
Figure 3. CCN activation curves of (NH4)2SO4 and NaCl particles under two experimental conditions
(∆T1 and ∆T2).
ρeff, χ and dve of Inorganic Salt Particles

Figure 4 shows an example mass distribution of DMA-selected NaCl particles
measured using the APM and CPC. As in the case of PSL, these distributions
were fitted by Gaussian functions to obtain the peak of the distribution, and then
we obtained the particle mass. ρeff, dve and χ were calculated from the experimen-
tal results.
Figure 4. Example mass distribution of NaCl particles prescribed by the DMA. The solid line is the fitting result
of the experimental data to a Gaussian function.
The calculated values are summarized in Fig. 5. ESA was used to calculate χ in
all cases, as χf and χc are not available. In the case of (NH4)2SO4, the measured
values of ρeff (1.67∼1.75 g cm−3) were slightly smaller than the bulk density
(1.77 g cm−3) (Fig. 5a), and χ was slightly larger for 6–60-nm (NH4)2SO4
particles of 1.02 to explain the dis-than unity (1.01∼1.04) (Fig. 5b). These values
are similar crepancy between theoretical calculations and experimental to those
obtained by Zelenyuk et al. (2006a), who showed results of the hygroscopicity
measurement. These results in-that the χ of (NH4)2SO4 is 1.03±0.01 at 160 nm
using a dicate that (NH4)2SO4 particles do not have a completely DMA and a
single-particle laser ablation time-of-flight mass spherical shape, as observed by
electron microscope (Dick spectrometer (SPLAT). Biskos et al. (2006a) estimated
a χ et al., 1998; Zelenyuk et al., 2006a).
Figure 5. ρeff, χ, and dve of (NH4)2SO4 and NaCl particles measured using the DMA-APM system. (a) ρeff of
(NH4)2SO4, (b) χ of (NH4)2SO4, (c) dve of (NH4)2SO4, (d) ρeff of NaCl, (e) χ of NaCl, and (f ) dve of NaCl
particles.
Comparing the results of RUN1 ∼ RUN4, systematic differences are observed.

ρeff was the highest in RUN2 and lowest in RUN4. These differences are greater
than the uncertainty of the ρeff measurement (1%). Zelenyuk et al. (2006b) re-
ported that rapid drying causes particles to have a more irregular shape. Thus, we
suspect that these differences found are possibly caused by different conditions of
the silica gel in the diffusion dryer in each run. Figure 5c shows the relationship
between dme and dve of (NH4)2SO4 particles. As can be easily expected from
the values of χ , the slope is almost equal to unity (0.99). Considering that the
error in the diameter of PSL particles is typically about 3% (Table 2), this result
shows that (NH4)2SO4 particles can be treated approximately as spherical in
most cases.
Figure 5d and e show ρeff and χ of NaCl particles, respectively. ρeff (1.75∼1.99
g cm−3) was smaller than the bulk density (2.16 g cm−3), and χ was significantly
larger than unity (1.05∼1.14). Kelly and McMurry (1992) used a DMA and an
impactor to measure ρeff of NaCl particles (dme=111 nm). They reported that
ρeff was equal to
1.86 g cm−3(χ=1.08). Zelenyuk et al. (2006a) measured ρeff of NaCl
(dme=160∼850 nm) using a DMA and a SPLAT. They showed that ρeff∼1.95 g
cm−3(χ∼1.06) at 160 nm.
Table 4. Values of dc_ve used for the calculation of S.
Mikhailov et al. (2004) estimated that χ is equal to 1.06 (99 nm) and 1.07
(201 nm) from hygroscopicity measurements. These values show that the shape of
NaCl particles is significantly different from spherical. Our results shown in Fig. 5
are similar to these other studies except for RUN4. In that case, χ was systemati-
cally higher than the other cases (1.10∼1.14). This value is higher than the χ for a
cubic shape in the continuous regime (1.08) (Hinds, 1999). Biskos et al. (2006b)
showed that χ calculated by ASA for a cubic shape is needed to explain hygro-
scopic growth of dme=6– 60-nm NaCl particles. χ is about 1.2 for dme=50–100
nm when we employ ASA to calculate the χ of cubic particles. Thus, we regard
the results of RUN4 as quite reasonable, considering that the shape of NaCl par-
ticles is cubic with rounded edges (Zelenyuk et al., 2006a). Figure 5f shows the
relationship between dme and dve of NaCl particles. dve is smaller than dme by
3–7%, and the value of the slope is 0.95. This shows that morphology should
be considered when using NaCl particles for calibration experiments (Rose et
al., 2008). The measurements of χ and ρeff without employing thermodynam-
ics models are limited to the size range of <150 nm (e.g., Kelly and McMurry,
1992). Our data are also useful for the investigation of the hygroscopic growth of
particles in this size range.
Calculation of S
In this section, we estimate S inside the CCNC using experimental results shown
in Sects. 4.1 and 4.2. In order to calculate volume equivalent critical dry diam-
eter (dc_ve), dc_me and ρeff at dc_me are required (Eq. 1). The dc_me shown in Sect.
4.1 was used, and ρeff at dc_me was calculated by interpolating the corresponding
experimental result of the DMAAPM system measurement (RUN3 and RUN4
for ∆T 1 and ∆T 2, respectively). The values of dc ve used for the calculation are
summarized in Table 4. The calculation was performed using the temperatures at

T 2 (Table 3).
Table 5 shows the calculated results. As discussed in Sect. 2.2, we regard the
model of Archer (1992) as the most reliable. Using this model, S values were
calculated to be 0.117% and 0.220% for ∆T 1 and ∆T 2, respectively. Thus, we
regard these values as the most probable values of S in the CCN chamber.
In the case of NaCl particles at ∆T 1, the value calculated using the approach
of Tang (1996) was the lowest, and a higher S value was obtained with the ap-
proach of Kreidenweis et al. (2005) and the ideal solution approximation.
Table 5. S values calculated using several thermodynamics models. See text for the input parameters and
descriptions of the models. “N/A” denotes that the thermodynamic data is not available.
The values calculated using the Pitzer models (Pitzer and Mayorga, 1973; Ar-
cher et al., 1992) are the same. For (NH4)2SO4 at ∆T 1, values similar to those
of Archer (1992) were obtained when we employed the Pitzer models (Pitzer and
Mayorga, 1973; Clegg et al., 1996). This indicates the validity of the Pitzer model
for the calculation of S. A similar value was obtained based on the work of Young
and Warren (1992). The ideal solution approximation for (NH4)2SO4 gave the
lowest S. This indicates the inappropriateness of the use of this approximation for
(NH4)2SO4. Parameterizations by Tang and Munkelwitz (1994) and Kreiden-
weis et al. (2005) estimated the highest values of S. These values are significantly
higher than those of Archer (1992). In the case of ∆T 2, the trend is similar to ∆T
1. For NaCl particles, aw given by Tang (1996) gave the lowest S, and the Pitzer
models (Pitzer and Mayorga, 1973; Archer, 1992) gave the highest S. In the case
of (NH4)2SO4, the ideal solution approximation gave the lowest values, and the
Pitzer models (Pitzer and Mayorga, 1973; Clegg et al., 1996) and that of Young
and Warren (1992) gave similar values. These trends are similar to that of Rose et
al. (2008). However, unlike the case of ∆T 1, a relatively large difference (0.005%
in absolute terms) between Clegg et al. (1996) and Archer (1992) was observed at
∆T 2. So far, we have been unable to determine what caused this difference.
To summarize the above discussion, the Pitzer models (Pitzer and Mayorga,
1973; Archer, 1992; Clegg, 1996) are valid to estimate critical S values within an
uncertainty of 2% (relative). The ideal solution approximation gives lower values
of the critical S, and the parameterizations by Tang and Munkelwitz (1994) and
Kreidenweis et al. (2005) yield higher values for (NH4)2SO4. These parameter-
izations are based on the data of hygroscopic growth up to RH=95%. The con-
centration of the (NH4)2SO4 solution at this RH is about m=1.5 mol Kg−1.
Figure 6 shows the φ of (NH4)2SO4 given by Clegg et al. (1996). In the case of
m<1 mol Kg−1, φ decreases with an increase in m because of the Debye Hückel
effect.
Figure 6. φ and related parameters for an aqueous solution of (NH4)2SO4 calculated based on the work of Clegg
et al. (1996). “Debye-Hückel term” is the first two terms in Eq. (B2), and “shortrange forces”corresponds to the
third and fourth terms in the same equation.
However, φ increases with the increase in m for m>2 mol Kg−1 due to short-
range forces. This shows that the thermodynamic properties of (NH4)2SO4
solutions change at around 1.5 mol Kg−1. Values of m at critical S values of
(NH4)2SO4 particles were calculated to be 0.01∼0.2 mol Kg−1(S=0.1∼1%), and
thus the parameterizations based on a concentration range of m>1.5 mol Kg−1
are not always appropriate to calculate critical S values because they overestimate
the magnitude of the non-ideality of the solution, which corresponds to an over-
estimation of critical S.
Summary and Conclusion

In this study, we measured CCN/CN ratios and mp of size-selected (NH4)2SO4
and NaCl particles. CCN/CN ratios were measured using a CCNC and a CPC,
and mp was measured using an APM and a CPC. The CCNC was operated under
two conditions (∆T 1 and ∆T 2). The dc_me values of (NH4)2SO4 were 115.2 nm
(∆T 1) and 75.3 nm (∆T 2). In the case of NaCl, these values were 94.7 nm (∆T
1) and 63.2 nm (∆T 2). dve calculated from mp and ρm were smaller than dme by
about 1 and 5% for (NH4)2SO4 and NaCl, respectively. Thus, it is a good approxi-
mation to treat (NH4)2SO4 particles as spherical particles.
dc_ve values were estimated from dc_me and measured ρeff, and they were
used as input parameters for Köhler theory calculations. Then, S values inside the
CCNC were estimated using several thermodynamics models. In these calcula-
tions, we regarded that the Pitzer model for NaCl (Archer, 1992) was the most
reliable, as it was in excellent agreement with the experimental data, including the
concentration range that is important for CCN activation. The values obtained
for (NH4)2SO4 and NaCl agreed to within a difference of 2% (relative) when
the Pitzer model was employed for the calculation. This result indicates that ap-
plication of the Pitzer model for calibrating a CCNC gives the most probable
values of S.
Appendix A
Table A1. List of parameters.
Appendix B
Thermodynamics Models for aw of Inorganic Salt
In this section, we review the thermodynamics models of (NH4)2SO4 and NaCl
employed in this study to estimate S in the CCNC.
B1 Ideal Solution Approximation

The simplest model is the ideal solution approximation (φ=1). Although it may
be too simple (e.g., Young and Warren, 1992), this approximation has been used
to calibrate CCNCs in many studies (e.g., Raymond and Pandis, 2002; Roberts
and Nenes, 2005). The manufacture of CCNC also relies on this model (Shilling
et al., 2007). Thus, it is important to compare this model with more sophisticated
models.
B2 Pitzer Model
Pitzer developed a semi-empirical model to calculate φ (Pitzer, 1973). Using the
original Pitzer model, φ of a single electrolyte can be calculated by the following
equation (see Appendix A for the notation of parameters):
3/2
1 2v v (2vmvx )
j = 1 - zm z x Af
1 + 1.2 I
(
(0)
+ m m x bMX
v
(1)
+ bMX )
exp (-a I ) + m 2
v
f
C MX (B1)
The first two terms are derived from Debye-Hückel theory, and the third and
fourth terms express short-range interactions (e.g., ion-molecular interactions). Aφ
can be calculated as a function of temperature using the polynomial equation giv-
en by Clegg et al. (1994), which is based on the study of Archer and Wang (1990).
(0) (1) f
Pitzer and Mayorga (1973) determined three parameters ( bMX , bMX and C MX )
for NaCl and (NH4)2SO4 using experimental data reviewed by Electrolyte Solu-
tions (Robinson and Stokes, 2002). The model has been used for the calibration
of CCNCs (e.g., Mochida et al., 2006) and compared with other models by Rose
et al. (2008).
Archer (1991) modified Eq. (B1) as follows:
I 2v v
ϕ = 1 − zm z x Aφ +m m x
1 + 1.2 I v
4vm2 v x
( (0)
bMX (1)
+ bMX )
exp (-a I ) + m 2
v
(C (0)
MX
(1)
+ C MX )
exp (-a2 I ) (B2)
Archer (1992) gave the parameters of the above equation for NaCl as a function
of temperature and pressure. Experimental data used by Archer (1992) cover the
concentration range that is important for the calculation of critical S (on the or-
der of 10−1∼10−2 m). Thus, we regard this model as giving the most reliable value
(Clegg, 2007). Note that the correction given by Clegg et al. (1994) should be
employed for the use of the work of Archer (1992). Clegg et al. (1996) obtained
parameters for Eq. (B2) for (NH4)2SO4, and their results have been used in some
CCN studies (e.g., Kumar et al., 2003), and it has been compared extensively
with other models by Kreidenweis et al. (2005).
B3 van’t Hoff Factor

Another method to express the non-ideal behavior of inorganic electrolyte solu-
tions is the use of i. One of the most widely used expressions for i was derived by
Young and Warren (1992):
i = 1.9242 − 0.1844 ln(m) − 0.007931(ln(m))2(B3)
This equation is applicable only to (NH4)2SO4. The value from this model has
been used in some studies (e.g., Svenningsson et al., 2006; Frank et al., 2007) and
compared with other models by Rose et al. (2008).
B4 Polynomial Equation
Tang and Munkelwitz (1994) and Tang (1996) measured hygroscopic growth of
(NH4)2SO4 and NaCl using the electrodynamic balance (EDB) technique. They
summarized the experimental results with the following polynomial function:
aw = 1.0 + å C i x i
(B4)
where Ci are constants, and x is the concentration of the solution in weight %.

This model has also been used to calibrate a CCNC (e.g., Snider et al., 2006)
and compared with other models by Kreidenweis et al. (2005) and Rose et al.
(2008).
Kreidenweis et al. (2005) fitted the growth factors (GFs) of (NH4)2SO4
and NaCl particles measured by a hygroscopic tandem DMA with the following
equation:
1/3
 a 
= 1 + ( a + baw + caw2 ) w  (B5)
d me _ wet
GF =
d me _ dry  1 + aw 
where a, b, and c are constants, and dme_wet and dme _dry denote the wet and dry par-
ticle diameters, respectively. This model has also been used for some CCN studies
(e.g., Mochida et al., 2006; Rose et al., 2008).
Appendix C
ρw and σ
Here we summarize values of ρw and σ employed for the calculation of critical
S. ρw depends both on temperature (Kell, 1975) and the concentration of solute
(Tang and Munkelwitz, 1994; Tang, 1996). However, the density of the solution
is available only at 25ºC, and the influence of the solute on the density of solution
near the critical Dp is estimated to be small. Thus, we employed the temperature-
dependent water density given by Kell (1975), which is written as follows:
5
å aT i
i
pw = i =0 (C1)
1 + a-1T
where ai are constants.
σ also depends on temperature and chemical composition (Seinfeld and Pan-
dis, 2006). We employed the following set of equations to calculate σ :
s(NH 4 ) SO4 = s _ water (T ) + 2.17 ´10-3 m( NH 4 )2 SO4

2
(C2)
σ NaCl = σ _ water (T ) + 1.62 ×10−3 mNaCl

(C3)
where
1.256
æT -T ö÷
swater (T ) = 235.8´10 ççç c -3
÷÷

è T ø÷ c
(C4)
Equations (C2) and (C3) are taken from Seinfeld and Pandis (2006) and Eq. (C4)
was given by Vargaftik et al. (1983).
Acknowledgements
This work was supported by the Ministry of Education, Culture, Sports, Sci-
ence, and Technology (MEXT) and the global environment research fund of the
Japanese Ministry of the Environment (B-083). M. Kuwata thanks to the Japan
Society for the Promotion of Science (JSPS) for a JSPS Research Fellowship for
Young Scientists. Edited by: G. Roberts
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Crystal Structure of
[Bis(L-Alaninato)Diaqua]
Nickel(II) Dihydrate
Awni Khatib, Fathi Aqra, David Deamer and Allen Oliver
Abstract
The title complex, [Ni(C3H6O2N)2(H2O)2]⋅2H2O, has been prepared from
nickel(II) chloride in aqueous solution by adding L-alanine and potassium
hydroxide. It has been crystallized from aqueous solution, and its structure
was determined by X-ray structure analysis. The nickel(II) ion adopts distort-
ed octahedral coordination geometry with two bidentate L-alanine molecules
and two water molecules. The complex is neutral and dihydrated. The crystal
structure shows the hydrogen bonding between water and amide hydrogens
within the lattice, and each fragment of the complex contains two water mol-
ecules as hydrated water. The L-alaninato ligand skeleton of the compound
adopts the most stable trans-III configuration in the solid state. The alternat-
ing two five-membered chelate rings are in the stable gauche conformation.
Introduction
Complexes formed by metal cations and organic species are incorporated in many
biochemical structures, such as cytochromes of mitochondrial membranes, hemo-
globin, and chlorophyll. Transition metal complexes with Schiff-base ligand con-
taining the carboxylate group have been of great interest due to their importance
as essentially biologically active [1–3] models for metalloproteins [4] and their
various geometry aspects [5]. Metals bound to amino acids are essential for the
catalytic function of certain enzymes, and their chemistry has received a great deal
of research interest due to their significant interaction with enzymes and withdif-
ferent organic ligands which enables a better understanding of the antitumor/viral
activities of this class of compounds and for modeling substrates involved in en-
zyme inhibition [6, 7]. A number of complexes of amino acids with many transi-
tion metal ions have been prepared and thoroughly studied [8–14]. A complex of
alanine with nickel(II) was reported [15] and described as a neutral bis(alaninato)
diaqua nickel(II), and its X-ray crystal structure seems not to have been explicitly
studied. Therefore, it was considered worthwhile and of great significant chemical
interest to synthesize this complex and to study thoroughly its crystal structure in
order to get greater depth into its composition.
Study of the structures of metal-amino acid complexes is a classical problem
initiated by the school of Pauling in the 40s, with the nickel-glycine compound.
Many authors have worked in this direction with increasing resolution and de-
tails. This paper describes the single-crystal structure of [bis(L-alaninato)diaqua]
Nickel(II) dihydrate.
Experimental
Chemicals and Instrumentation
All chemicals were of reagent grade and used as purchased from commercial
source. The single-crystal X-ray diffraction data in this paper were recorded on
an instrument Bruker APEX-II 3-circle diffractometer, a CCD area detector with
graphite monochromated Mo-Kα radiation supported by the National Science
Foundation, Major Research Instrumentation (MRI) Program under Grant no.
CHE-0521569.
Preparation Procedures
NiCl2⋅6H2O (20 mL, 0.1 M), KOH (20 mL, 1.0 M), and L-alanine (20 mL,
0.2 M) were mixed. The mixture was made basic with pH = 8 and turned from
Crystal Structure of [Bis(L-Alaninato)Diaqua]Nickel(II) Dihydrate 265
green to pale blue. The flask solution was left at room temperature. After standing
for two weeks, pale-blue tablet-shaped crystals were obtained, removed, and dried
under vacuum. The isolated crystals were subjected to X-ray studies.
Crystal Structure Determination

The structure was solved by direct methods and expanded routinely. The model
was refined by full-matrix least-squares analysis of F2 against all reflections. All
nonhydrogen atoms were refined with anisotropic thermal displacement param-
eters. The hydrogens on the water oxygens and amide nitrogens were located from
a difference Fourier map and included in their observed positions with thermal
parameters tied to that of the atoms to which they are bonded. The hydrogen
atoms bonded to carbon were included in calculated positions with thermal pa-
rameters tied to that of the carbon to which they are bonded. The crystallographic
data and parameters are given in Table 1. The softwares used for direct method,
least-squares analysis, molecular drawing, and preparing the crystallographic ma-
terials are APEX-II [16], SAINT [17], XPREP [18], SADABS [19], SHELXTL
[20], and ORTEPII [21].
Table 1. Crystal data and structure refinement of the title compound.


The title complex was prepared by the reaction of NiCl2⋅6H2O, KOH, and L-
alanine. The presence of KOH has two roles: the first is to adjust the pH of the
resulting solution from 6 to 8 and the second is to convert the carboxylic group of
alanine to carboxylate ion in favor to bind readily with nickel(II) ion.
There are four molecules of the nickel complex and eight water of crystalliza-
tion in the unit cell of the C-centered, acentric, and monoclinic space group C2.
The correct enantiomorphof the space group and handedness of the molecule
were determined by comparison of the intensities of Friedel pairs of reflections
(Flack parameter= 0.018(13)) and by the known stereochemistry of the L-alanine
ligands. Both techniques agreed, and the correct configuration is shown in the
Figure 1.
Figure 1. The chemical diagram and the crystal structure of the title compound showing the atomic numbering
scheme.
The title compoundis crystallized from aqueous solution as pale-blue prismat-

ic crystals. The structure consists of an [Ni(L-alaninato)2(H2O)2] and two water
molecules. The nickel ion resides at the center of symmetry of the octahedron
and is surrounded by two oxygen atoms of two alanine molecules, two oxygen
atoms of two water molecules, and two nitrogen atoms of the same two alanine
molecules. The carboxylato oxygens and the amido nitrogens of the two alanine
molecules define the equatorial positions, whereas the two oxygen atoms of the
two water molecules occupy the axial ones. It is observed that the axial Ni–O
bond distances (Table 2) of 2.0706(18) and 2.1006(16) Å are significantly longer
than the equatorial Ni–O bonds of 2.0422(17) and 2.0567(17) Å. All the Ni–O
distances are in agreement with those found in six coordinate nickel(II) complexes
[22]. The average Ni–N bond distance of 2.073(17) Å is in the normal range for
Ni–N primary amines of high-spin octahedral nickel(II) complexes with chelat-
ing ligands [23].
The axial angle O–Ni–O is 178.36(9)°, whereas the equatorial O–Ni–O is
179.33(7)° (Table 2) that are close to linearity. The average Ni–O and Ni–N
bond lengths are in accordance to that known for nickel(II) distorted octahedral
geometry. Therefore, two alanine molecules and two water molecules are directly
involved in coordination. The coordination geometry around the nickel(II) ion
is a six-coordinated tending toward distorted octahedral, with a metal center not
lying exactly within the N2O2 plane because the bond angles are not perfect [24,
25]. The two apical positions are occupied by water molecules, and the equatorial
plane is occupied by the chelating alanine ligands. Distortions about nickel atom
are observed, in which slightly different bond distances to the coordinating water
molecules; essentially identical bond distances to the nitrogens and slightly differ-
ent bond distances to the chelating oxygens O1 and O3. The enforced distortion
about the equatorial plane due to the formation of the two five-membered chelate
rings is seen. The two ligands adopt an envelope and a planar geometry, with re-
spect to the mean equatorial plane about the nickel (Table 3).
Table 2. Important bond lengths [Å] and angles [°] of the title compound.
Table 3. Deviation from the mean plane.
It is seen that there are two water molecules not chemically bonded to Ni(II)
and located at the opposite site of alanine group, and have no significant interac-
tion with the metal atom. A hydrogen bonding is observed between the hydrogen
atoms of coordinated and hydrated waters with the oxygen atoms of the carboxy-
lato groups, there are many hydrogen bonds responsible of the packing, and the
values of these interactions are shown in Table 4. Also, the hydrogen bonds are
seen between the hydrogen of the amide nitrogen and the oxygen atoms of the
hydrated water molecules and carboxylato groups (Figure 2). The hydrogen atoms
on the water molecules and the amide nitrogens were all located from a differ-
ence Fourier map. All are involved in an extensive three-dimensional network of
hydrogen bonds within the lattice.
Table 4. Hydrogen bonds of the title compound [Å and °].

Figure 2. Hydrogen bonding network of the title compound.
Conclusions
This communication describes the crystallographic characterization of a complex
of nickel(II) with L-alanine. The method illustrated for the preparation of this
complex must be extended to other metal ions such as iron, copper, and zinc. In
fact, nickel(II) was chosen for our synthesis because it forms well-defined crystals
that can be studied by X-ray crystallography. The complex is a chelate with two
bidentate alanine ligands bonding through N and O and two water molecules.
The oxygen of the carboxylato groups of alanine is deprotonated by removal of its
hydrogen with the hydroxyl group of KOH producing water molecule.
Supplementary Material
Crystallographic data for the structure reported in this paper have been deposited
at the Cambridge Crystallographic Data Centre (CCDC) and allocated deposi-
tion no. CCDC 718341 for the title compound and can be obtained free of
charge on application to CCDC 12 Union Road, Cambridge CB2 1EZ, UK (Fax:

(internat.) 44(1223)336-033; E-mail:deposit@ccdc.cam.ac.uk).
Acknowledgements
The authors would like to thank Mohammad Zhour, the university web master
for computer assistance. Also, A. Khatib wishes to thank the CIES and Fulbright
for a Sabbatical at the University of California.
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Mean Amplitudes of Vibration
of the IF8 − Anion
Enrique J. Baran
Abstract
The mean amplitudes of vibration of the interesting IF8 − anion (D4d-sym-
metry), containing iodine (VII), were calculated from known spectroscopic
and structural data in the temperature range between 0 and 1000 K. The re-
sults are discussed in comparison with those of related species.
Introduction
Mean amplitudes of vibration are very useful and valuable parameters for the
analysis of molecular structures and their vibrational behavior. In a similar way to
vibrational frequencies and force constants, they can be very characteristic values
for both bonded and nonbonded atoms [1, 2].
During years we have calculated mean amplitudes of vibration for a large se-
ries of molecules and ions containing halogen-halogen or halogen-oxygen bonds
(for a recent review cf. [3]) and in this paper we present the results of our calcula-
tions for the interesting IF8− anion, which vibrational-spectroscopic behavior was
only very recently definitely clarified [4].
As it is well known, the structure of this anion is a practically perfect Archi-
medean square antiprism [5], constituting the unique example of an interhalogen
species presenting this geometry, which structural peculiarities are similar to those
of the few other known examples of homoleptic species of this type, namely,
ReF82−, ReF8−, WF82−, UF82−, and XeF82− [6, 7].
Calculations
The mean amplitudes of vibration were calculated with the method of the “char-
acteristic vibrations” of Müller et al. [8] (cf. also [2, 9]). The necessary vibrational-
spectroscopic data were taken from the recent paper of Dixon et al. [4] and the
geometrical parameters from the paper of Mahjoub and Seppelt [5].

The obtained results, in the temperature range between 0 and 1000 K, are shown
in Table 1. Regarding the nonbonded F. . .Fpairs, F. . .F (in plane) refers to the
pairs within one hemisphere of the anion, whereas F. . .F (betw. planes) refers to
neighboring pairs belonging to different hemispheres.
Table 1. Mean amplitudes of vibration (in Å) for the IF8− anion in the temperature range between 0 and
1000 K.
Mean Amplitudes of Vibration of the IF8 − Anion 275
The analysis of the so far available data of mean amplitude values for I–F
bonds has shown that the extreme values lie between 0.0377 Å (for IF6+) and
0.0602 Å (for IF52−) [3, 10], in agreement with the fact that in the first case io-
dine presents the oxidation state +7 and a positive charge whereas in the other one
the iodine is in the oxidation state +3 and not only presents two negative charges
but also an important congestion effect on the molecular plane, in which the fluo-
rine atoms are practically in contact [3, 10, 11]. Besides, these two species present
also the greatest differences in bond lengths found in I/F species (1.75 Å for IF6+,
2.095 Å for IF52−) [3, 12]. Furthermore, the specially high mean amplitude value
of IF52− is in good agreement with the very low force constant calculated for the
I–F bonds in this anion (1.53 mdyn/Å [11]).
The values of the mean amplitudes of vibration calculated for the I–F bonds
of IF8− fall clearly into the mentioned range as it can also be seen from the com-
parative data presented in Table 2. This comparison shows that the values for
IF8− are appreciably higher than those found for IF6+ showing again the effect of
the geometry and of the negative charge over bond weakening [3]. Besides, these
amplitude values are only somewhat higher than those calculated for the equato-
rial IF7 bonds.
Table 2. Mean amplitudes of vibration (in Å) of some iodine (VII) species at three different temperatures ((eq):
equatorial I–F bonds; (ax): axial I–F bonds).
On the other hand, the values calculated for IF8− lie relatively close to those of
the equatorial I–F-bonds of IOF6−. In comparison with the interhalogen bonds
of the other two fluorooxoanions containing iodine (VII), IF8− presents lower
mean amplitudes of vibration (i.e., stronger I–F bonds) than IO2F52− but weak-
er I–F bonds than IO2F4−, in the full temperature range.
Concerning the amplitude values of the nonbonded pairs, those of the same
hemispheres are always lower and show a smaller temperature dependence than
those between F-atoms belonging to the different hemispheres.
Conclusions
Mean amplitudes of vibration of the IF8− anion clearly lie in the expected range
determined for I–F bonds. These values point to relatively weak bonds, when
compared with iodine fluorine bonds present in other simple iodine (VII) species,
such as IF7 or IF6+, in agreement with the higher coordination number and with
the presence of a negative charge in the case of the IF8− anion.
Acknowledgements
This work has been supported by the Consejo Nacional de Investigaciones
Científicas y Técnicas de la República Argentina (CONICET) and the Univer-
sidad Nacional de La Plata. The author is a member of the Research Career of
CONICET.
References
1. S. J. Cyvin, Molecular Vibrations and Mean Square Amplitudes, Elsevier,
Amsterdam, The Netherlands, 1958.
2. A. Müller, E. J. Baran, and K. H. Schmidt, “Characteristic mean amplitudes of
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Mechanistic Aspects of
Osmium(VIII) Catalyzed
Oxidation of L-Tryptophan
by Diperiodatocuprate(III) in
Aqueous Alkaline Medium:
A Kinetic Model
Nagaraj P. Shetti, Ragunatharaddi R. Hosamani

and Sharanappa T. Nandibewoor
Abstract
In presence of osmium(VIII), the reaction between L-tryptophan and
diperiodatocuprate(III) DPC in alkaline medium exhibits 1:4 stochiom-
etry (L-tryptophan:DPC). The reaction shows first-order dependence on
[DPC] and [osmium(VIII)], less than unit order in both [L-tryptophan] and
Mechanistic Aspects of Osmium(VIII) Catalyzed Oxidation 279
[alkali], and negative fractional order in [periodate]. The active species of

catalyst and oxidant have been identified. The main products were identi-
fied by spectral studies and spot test. The probable mechanism was proposed
and discussed.
Introduction
In the recent past [1], some relatively stable copper (III) complexes have been
prepared, namely, the periodate, guanidine, and tellurate complexes. The Cu3+/
Cu2+ reduction potential is –1.18 V in alkaline solution [2]. The copper(III) pe-
riodate complex (DPC) exhibits different multiple equilibria involving different
copper(III) species in aqueous alkaline medium. It is interesting to know which
of the copper(III) species is the active oxidant.
L-tryptophan (L-trp) is an essential aminoacid and it is needed to maintain
optimum health. Osmium(VIII) acts as an efficient catalyst in many redox reac-
tions [3, 4] involving different complexities due to the formation of different
intermediate complexes, free radicals, and multiple oxidation states of osmium.
The uncatalyzed reaction of oxidation of L-tryptophan by DPC has been stud-
ied [5]. We have observed that the microamounts of osmium(VIII) catalyze the
oxidation of L-trp by DPC in alkaline medium. In order to understand the active
species of oxidant and catalyst and to propose the appropriate mechanism, the
title reaction is investigated in detail, in view of various mechanistic possibilities.
Experimental
All chemicals used were of reagent grade and millipore water was used through-
out the work. A solution of L-trp (s.d. fine) was prepared by dissolving an ap-
propriate amount of recrystallized sample in millipore water. A stock solution of
osmium(VIII) was prepared and standardized by the method reported earlier [6].
The copper(III) periodate complex was prepared [7] and standardized by standard
procedure [8].
Kinetic Measurements
All kinetics measurements were carried out as in earlier work [6].

The results indicated 1:4 stoichiometry as given in Scheme 1.
Scheme 1. 1:4 stochiometry of osmium(VIII) catalyzed oxidation of L-trp by DPC reaction.
The main product, indole-3-acetic acid, was separated by TLC, using the mix-
ture of methyl acetate, isopropanol, and 25% ammonium hydroxide in the ratio
of 45:35:20. IR, NMR spectra and its melting points were compared with the
literature and were in good agreement. The LC-MS analysis of isolated product
indicated the presence of indole-3-acetic acid as molecular ion peak, m/z 175.
In the presence of catalyst, the reaction is understood to occur via parallel
paths with contributions from the uncatalyzed and catalyzed paths. The total rate
constant (kT) is equal to the sum of the rate constants of the catalyzed (kC) and
uncatalyzed (kU) reactions. Hence, kC=kT−kU. The reaction orders have been
determined from the slopes of log kc versus log (concentration) plots by varying
the concentration of L-trp, Os(VIII), OH−, and IO4−, in turn, while keeping
the other concentrations constant. The order in both [DPC] and [Os(VIII)] was
found to be unity. The order in [L-trp] and [OH−] was found to be less than
unity, and in [periodate] to be negative and less than unity. It is well known that
[9] Os(VIII) exists as (OsO4(OH)2]2+ in aqueous alkaline medium. It was found
that the increase in ionic strength increased the rate of reaction and decrease in
dielectric constant of the medium increased the rate of reaction. Initially added
products did not have any significant effect on the rate of reaction. Test for free
radicals indicated the participation of free radical in the reaction [6]. These experi-
mentally determined orders and results could be well accommodated in Scheme 2.
Based on the experimental results, monoperiodatocuprate MPC was consid-
ered to be the active species of DPC complex. The fractional order with respect
to L-trp concentration indicates the formation of a complex between L-trp and
osmium(VIII) species. Spectroscopic evidence for the complex formation between
catalyst and substrate was obtained from UV-vis spectra of Os(VIII), L-trp, and
a mixture of both. A bathochromic shift of about 6 nm from 255 nm to 261 nm
in the spectra of Os(VIII) was observed. The Michaelis-Menten plot also proved
the complex formation between catalyst and reductant. Such a complex between
a substrate and a catalyst has been observed in other studies [6].
Scheme 2. The osmium(VIII) catalyzed oxidation of L-trp by DPC.
Scheme 2 leads to the following rate law:
Rate
= kC = kT - kU
[ DPC ]

kK 1K 2 K 3 [ L - trp ] éëOH - ùû éëOs (VIII )ùû (1)
=
Â
where  denotes [H3IO63−]+K1[OH−][H3IO63−]+K1K2[OH−]+K1K2K3[OH−][L-

trp] which explains all the observed kinetic orders of different species. The rate law
(1) can be rearranged into the following form which is suitable for verification:
éOs (VIII )ù é H 3 IO6 3- ù é H 3 IO6 3- ù 1 1

ë û= ë û + ë û + + . (2)
kC é -ù
kK 1K 2 K 3 [ L - trp ] ëOH û kK 2 K 3 [ L - trp ] kK 3 [ L - trp ] k
Figure 1. Verification of rate law (1) of Os(VIII) catalyzed oxidation of L-tryptophan by DPC at 298 K
(conditions as in Table 1). (a) [Os(VIII)]/kc versus 1/[L-trp]; (b) [Os(VIII)]/kc versus 1/[OH−]; (c) [Os(VIII)]/
kc versus [H2IO63−].
According to (2), others being constant, the plots of [Os(VIII)]/kC versus 1/[L-
trp], [Os(VIII)]/kC versus 1/[OH−], and [Os(VIII)]/kC versus [H3IO62−] were
linear as in Figure 1. From the intercepts and slopes of such plots, the reaction
constants K1, K2, K3, and k were calculated as (15.6±0.4) dm3 mol−1, (3.3±0.10)
x 10−4 mol dm−3, (0.71±0.02) × 104 dm3 mol−1, (3.2±0.04) × 103 dm3 mol−1s−1,
respectively. The values of K1 and K2 obtained were in good agreement with previ-
ously reported values [10]. These constants were used to calculate the rate con-
stants over different experimental conditions; when compared with the experi-
mental kC values, they were found to be in reasonable agreement with each other,
which fortifies Scheme 2.
Similarly K1, K2, K3, and k were calculated at four different temperatures
(288, 293, 298, and 303 K) and used to compute the activation parameters and
thermodynamic quantities. The values of Ea, ΔH#, ΔS#, and ΔG# and log A were
obtained and found to be 42.0 ± 2 kJ mol−1, 44.0 ± 2 kJ mol−1–30.0±1.5 J K−1
mol−1, 53.0 ± 3 kJ mol−1, and 11.0±0.5, respectively. (Ea, ΔS#, ΔH#, and log
A were 51.7 ± 3 kJ mol−1, −114 ± 6 J K−1 mol−1, 48.2 ± 2 kJ mol−1, and 10.5,
resp., for the uncatalyzed reaction [5].) The catalyst Os(VIII) alters the reaction
path by lowering the energy of activation (i.e., it provides an alternative pathway
with lower activation parameters for the reaction).
The thermodynamic quantities, ΔH (kJ mol−1), ΔS (J K−1 mol−1), and ΔG
(kJ mol−1) using K1 were calculated to be –47, 182, and −6.4, respectively. Simi-
larly the values using K2 were calculated to be 97.7, 262.8, and 18.6, respectively
and the values using K3 to be –144.0, −412.0, and −22.0, respectively.
The effect of ionic strength and dielectric constant of the medium on the rate
explains qualitatively the reaction between two negatively charged ions, as seen in
Scheme 1. The moderate ΔH# and ΔS# values are favorable for electron transfer
reaction. The negative value of ΔS# suggests that the intermediate complex is
more ordered than the reactants [11]. The observed modest enthalpy of activation
and a higher-rate constant for the slow step indicate that the oxidation presum-
ably occurs via an innersphere mechanism. This conclusion is supported by earlier
observations [12].
Conclusion
Among various species of Cu(III) in alkaline medium, monoperiodatocuprate(III)
is considered to be the active species for the title reaction. The active species of
osmium(VIII) is understood to be as [OsO4(OH)2]2−. The activation parameters
evaluated for the catalyzed and uncatalyzed reactions explain the catalytic effect
on the reaction. The Os(VIII) catalyst alters the reaction path by lowering the
energy of activation.
Table 1: Effects of [DPC], [L-trp], [OH−], [IO4−], and [Os(VIII)] on the osmium(VIII) catalyzed oxidation of
L-trp by DPC in alkaline medium at 298 K, I = 0.20 mol dm−3.
References
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Kinetic and Mechanistic
Studies on the Reaction of
DL-Methionine with [(H2O)
(tap)2RuORu(tap)2(H2O)]2+
in Aqueous Medium at
Physiological pH
Tandra Das A. K. Datta and A. K. Ghosh
Abstract
The reaction has been studied spectrophotometrically; the reaction shows two
steps, both of which are dependent on ligand concentration and show a lim-
iting nature. An associative interchange mechanism is proposed. Kinetic and
activation parameters (k1 ∼10−3s−1 and k2 ∼10−5s−1) and (ΔH1≠ = 13.8 ±
1.3 kJmol−1, ΔS1≠ = −250 ± 4JK−1 mol−1, ΔH2≠ = 55.53 ± 1.5kJ mol−1,
Kinetic and Mechanistic Studies on the Reaction 287
and ΔS2≠ = −143 ± 5JK−1mol−1) have been calculated. From the temperature
dependence of the outer sphere association equilibrium constant, thermody-
namic parameters (ΔH1° = 16.6 ± 2.3kJmol−1 and ΔS1° = 95 ± 7JK−1mol−1;
ΔH2° = 29.4 ± 3.2kJmol−1 and ΔS2°= 128 ± 10JK−1mol−1) have also been
calculated.
Introduction
The binding of the antitumor drug cisplatin and other platinum group metal
complexes, especially ruthenium(II), rhodium(III), iridium(III), platinum(II),
and palladium(II) to amino acids, nucleosides, nucleotides, and particularly to
DNA is still an interesting subject and has given considerable impetus to research
in the area of metal ion interactions with nucleic acid constituents. Ruthenium
complexes are an order of magnitude less toxic than cisplatin, and aqua complexes
if used directly will be less toxic as some hydrolyzed side products are responsible
for toxicity. From a literature survey [1–3], it is revealed that many potential alter-
native metallopharmaceuticals have been developed, ruthenium being one of the
most promising, and are currently undergoing clinical trials [4–7]. Another point
of interest is that DNA is not the only target. Binding to proteins, RNA [8–10]
and several sulphur donor ligands, present in the blood, are available for kinetic
and thermodynamic competition [11, 12].
Keeping this in mind, in this paper, we have studied the kinetic details of the
interaction of our chosen complex (an aqua-amine complex of ruthenium(II))
with an S-containing amino acid DL-methionine at pH 7.4 in aqueous medium
and a plausible mechanism is proposed.
The importance of the work lies in the fact that (a) the reaction has been stud-
ied in an aqueous medium, (b) the reaction has been studied at pH (7.4) which is
the physiological pH of the human body, (c) the aqua-amine complex is chosen,
(d) ruthenium(II) than ruthenium(III) is chosen, as ruthenium(III) is a prodrug
which is reduced in the cell to ruthenium(II), and (e) the title complex maintains
its +2 oxidation state even at pH 7.4 due to the presence of a strong pi-acceptor
ligand tap (tap={2-(m-tolylazo)pyridine}), where most of the other ruthenium(II)
complexes are oxidized to ruthenium(III).
Materials and Methods

Reported method [13, 14] was used to isolate cis-[Ru(tap)2(H2O)2](CIO4)2⋅H2O.
The reacting complex ion [(H2O)(tap)2RuORu(tap)2(H2O)]2+ (1) was generated
in situ by adjusting the pH at 7.4. The reaction product [(tap)2Ru(μ-O)(μ-meth)
Ru(tap)2]2+ (complex 2) of DL-methionine, and complex 1 is shown in Figure 1.

The composition of 2 in solution was determined by Job’s method of continuous
variation and the metal: ligand ratio was found to be 2:1. The pH of the solution
was adjusted by adding NaOH/HClO4, and the measurements were carried out
with the help of a Sartorius make digital pH meter (PB 11) with an accuracy of
±0.01 unit.
Doubly distilled water was used to prepare all the kinetic solutions. All chemi-
cals used were of AR grade, available commercially. The reactions were carried out
at constant ionic strength of (0.1 M NaClO4).
Figure 1. Difference in spectrum between complex 1 and product complex (2); [1] = 1.0×10−4 mol dm−3, [DL-
methionine] = 2.0×10−3 mol dm−3, cell used 1 cm quartz.
Kinetics
The kinetic studies were done on a Shimadzu UV-2101PC spectrophotometer
attached to a thermoelectric cell temperature controller (model TB 85, accuracy
±0.1°C). The progress of the reaction was monitored by following the decrease
in absorbance at 600 nm using mixing technique and maintaining pseudo-first-
order conditions. In Figure 2, plot of ln(At−A∞) versus time shows a consecutive
nature of the reaction. Initially, it is curved and shows linear behavior in the latter
stage. The rate constants were calculated using the method of Weyh and Hamm
[15] as described in an earlier paper [1] using the following equation:
lnΔ = constant − k1(obs) t, when t is small. (1)

The meaning of Δ is shown in Figure 2 (Δ = X − Y). k2(obs) is calculated from

the latter linear portion.
Figure 2. A typical plot of ln(At−A∞) versus time.

At a fixed excess [DL-methionine] (2.0 × 10−3mol dm−3), pH 7.4, temperature
50°C, and ionic strength (0.1mol dm−3 NaClO4) the reaction was found to be first
order in [complex 1], that is, d [complex 2]/dt=kobs [complex 1].
The pKa1 and pKa2 values [16] of DL-methionine are 2.24 and 9.07, re-
spectively, at 25°C. Thus, at pH 7.4, the ligand exists mainly as a neutral mol-
ecule, that is, as a zwitterion (LH2+→LH→L−). On the other hand, first acid
dissociation equilibrium of the complex [Ru(tap)2(H2O)2]2+ is 6.6 [17] at
25°C. At pH 7.4, the complex ion exists in dimeric oxo-bridged form, [(H2O)
(tap)2RuORu(tap)2(H2O)]2+ [18–21]. At pH 7.4, the mononuclear species ex-
ists in the hydroxoaqua form. Two such species assemble to form the dinuclear
oxo-bridged diaqua complex due to thermodynamic force mainly arising from
pi-bonding [22] (O2− donor, RuII acceptor) which is favorable for 4d ion, RuII.
Now, such strong covalency reduces the acidity of the coordinated water. The
oxo-bridge formation is solely dependent on pH. Electrochemical studies show
that there is pH potential domain, where the μ-oxo structures stay intact. Variable
temperature study does not show any effect, which is in line with the fact that
oxo-bridge formation is solely pH-dependent [23, 24]. The rate constant for such
process can be evaluated by assuming the following scheme
(1) 
k1
→ B 
k2
→(2), (2)
where B is [(H2O)(tap)2RuORu(tap)2(LH)]+.
Calculation of k1 and k2 Values for Step (1) → B and for

(B) → (2) Step
The rate constants, k1(obs) for (1) → B and k2(obs) for (B) → (2), were calculated
following the technique described in an earlier paper [25], and the values are
collected in Tables 1 and 2. The rate increases with the increase in [ligand] and
reaches a limiting value for both steps. Details of the mechanism are discussed in
“Mechanism and Conclusion” section. The k1, k2, KE′, and KE′′ for the two steps
are calculated similarly and collected in Table 3.
Table 1. 103k1(obs) values for different ligand concentrations at different temperatures. [Complex] = 1 × 10−4mol
dm−3, pH = 7.4, ionic strength = 0.1mol dm−3 NaClO4.
Table 2. 105k2(obs) values for different ligand concentrations at different temperatures.
[Complex] = 1 × 10−4mol dm−3, pH = 7.4, ionic strength =0.1 mol dm−3 NaClO4.
Table 3. The k1, KE′, k2, and KE′′ values for the interaction of methionine with (1).
Effect of Change in pH on the Reaction Rate

This was studied at five different pH values. 103k1(obs)(s−1) and 105k2(obs) values are
0.73, 0.76, 0.83, 1.04 and 1.55 (s−1), and 3.3, 3.7, 4.16, 6.6, and 11.32 (s−1) at
pH 5.5, 6.0, 6.5, 7.0, and 7.4, respectively. In the studied pH range (pH 5.5 to
7.4), the percentage of diaqua species is reduced with the increase in pH, and the
percentage of the dimer is predominant. The dimer with its two metal centers is a
better center to the incoming nucleophiles. On the other hand, the pK1 and pK2
values of the ligand DL-methionine are 2.24 and 9.07 at 25°C. With the increase
in pH from 5.0 to 7.4, the amount of the deprotonated form increases, and the
zwitterionic form (LH) predominates which also partly accounts for the enhance-
ment of the rate with increase in pH.
Effect of Temperature on the Reaction Rate

Four different temperatures with varied ligand concentrations were chosen, and
the results are listed in Tables 1 and 2. The activation parameters for the steps (1)
→ B and (B) → (2), evaluated from the linear Eyring plots and compared with
the analogous systems [1], support the proposition.
Mechanism and Conclusion

The low ΔH≠ value, together with negative ΔS≠ value, suggests ligand participa-
tion in the transition state, and an associative interchange mechanism is proposed
(Scheme 1) for the interaction of DL-methionine with the title complex. The
bonding mode of methionine is not fully understood, as it was not possible to
isolate the solid product. In the studied reaction condition, that is, at pH 7.4,
methionine exists in the deprotonated form. At first S attacks on one of the two
ruthenium(II), centers are assumed. This step is ligand dependent, and with in-
creasing the ligand concentration, a limiting rate is reached. This may be due to
the formation of outersphere association complex, which is possibly stabilized
through hydrogen bonding. The spontaneous formation of an outersphere asso-
ciation complex is also supported from a negative ΔG° value calculated from the
temperature dependence of the KE values. The corresponding thermodynamic

parameters are ΔH1° = 16.6 ± 2.3 kJ mol−1 and S1° = 95 ± 7 JK−1 mol−1, ΔH2° =2
9.4 ± 3.2 kJ mol−1 and ΔS2° = 128 ± 10 JK−1 mol−1.
Scheme 1
The coordinated methionine in any of the ruthenium(II) centers now attacks

the second ruthenium(II) center like a metalloligand, and we observe two distinct
ligand dependent steps. For the ligand to behave as a bridging ligand with the
oxo-bridging complex, the mono atom sulphur [26, 27] bridging has the best
prospects. It is to be noted here that the second step is not a normal cyclisation
step as occurs in chelation in a single central atom. Here, two metal centers are
available, and after attachment of the ligand to one of the metal centers, the en-
vironment of the two centers will no longer remain the same, and when the dif-
ference in rate between two steps is larger, we observe the dependence of rate on
ligand concentration carried to the second step. But when the difference between
two steps is comparatively smaller as is found in a system earlier [2], the second
step is found to be independent on ligand concentration. A plausible mechanism

is shown here to commensurate with the experimental findings.
Acknowledgement
The authors would like to acknowledge The University of Burdwan, West Bengal,
India for assistance throughout the entire work.
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Molybdenum and
Tungsten Tricarbonyl
Complexes of Isatin with
Triphenylphosphine
M. M. H. Khalil and F. A. Al-Seif
Abstract
Reaction of M(CO)6; M = Mo or W with isatin in the presence of triphe-
nylphosphine in THF under reduced pressure gave the tricarbonyl derivatives
complexes [M(CO)3(isatH)(PPh3)]. The two complexes were characterized
by elemental analysis, infrared, mass and 1H NMR spectroscopy. The spectro-
scopic studies show that the two complexes exist in fac- and mer-isomers in so-
lutions through exchange the CO group and PPh3. The Uv-Vis spectra of the
complexes in different solvents were studied.
Molybdenum and Tungsten Tricarbonyl Complexes 297
Introduction
Isatin (2,3-dihydroindole-2, 3-dione) is a versatile lead molecule for designing
potential bioactive agents, and its derivatives were reported to possess broad-
spectrum antiviral activity [1, 2]. In the previous reports, the synthesis and char-
acterization of group 6 and 8 complexes of isatin and 5-methylisatin in absence
and presence of bipyridine were investigated [3, 4]. In this article, we report the
synthesis and characterization of molybdenum and tungsten complexes of isatin
in the presence of PPh3. The aim of these reactions is the synthesis and study of
mixed-ligand complexes, where the metal is surrounded by different donor atoms
in the coordination sphere, that is, the oxygen from isatin and phosphorous atom
from the triphenylphosphine (PPh3). PPh3 is different from the carbonyl group
since it is a strong σ-donor and weak π-acceptor ligand. Furthermore, the organic
phosphenes increase the stability of the transition metal complexes in the low-
oxidation state. Taking into account the electronic spectra the combination of a
reducing metal and an acceptor ligand generates a metal-to-ligand charge transfer
(MLCT) excited state which may appear in absorption and emission [5, 6].
Experimental
Reagents
Mo(CO)6, W(CO)6, isatin, and PPh3 were supplied from (Sigma Aldrich, St.
Louis, USA). All the solvents were reagent grade and purified prior to use.
Instruments
IR measurements were recorded as KBr pellets on a Unicam-Mattson 1000 FT-IR
spectrometer. Electronic absorption spectra were measured on a Unicam UV2-
300 UV-vis spectrophotometer. 1H-NMR measurements were performed on a
Varian-Mercury 300 MHz spectrometer. Samples were dissolved in (CD3)2SO
with TMS as internal reference. The complexes were also characterized by elemen-
tal analysis (Perkin-Elmer 2400 CHN elemental analyzer) and mass spectroscopy
(Finnigan MAT SSQ 7000). Table 1 gives the elemental analyses and mass spec-
trometry data for the complexes.
Table 1. Elemental analysis and mass spectrometric data for the molybdenum and tungsten complexes.
[Mo(CO)3(isatH)(PPh3)] Mo(CO)6 (0.20 g; 0.76 mmol), isatin (0.06 g;

0.33 mmol), and PPh3 (0.09 g; 0.33 mmol) were mixed in ca 30 ml tetrahydro-
furan. The mixture was degassed and heated to reflux for 4 hours, where the color
of the solution changed from yellow to dark red. The reaction mixture was cooled
and the solvent was removed under vacuum. The obtained solid was washed sev-
eral times with hot benzene and petroleum ether to give brown crystals with a
yield of 55% based on the metal.
[W(CO)3(isatH)(PPh3)] A similar procedure was performed as in the case
of [Mo(CO)3(isatH)(PPh3)] but the reaction time was 11 hours (reddish brown
powder, yield 48%).

IR and NMR Studies
Reactions of M(CO)6; M=Mo or W with isatin in the presence of PPh3 result-
ed in the formation of [M(CO)3(isatH)(PPh3)] complexes. The IR spectra of
the complexes exhibited characteristic bands of the isatin and PPh3 ligands with
the corresponding shifts, Table 2. In addition, the IR spectra of the complexes
showed that the νCO of isatin ligand exerted 20–45 cm−1 shift to lower frequency
suggesting that the coordination of isatin occurred in the range of ketoform in
both complexes. On the other hand, the IR spectra of the two complexes ex-
hibited three bands in the metal terminal carbonyl region [7] with shifts toward
the low-frequency region, Table 2. Also, the IR spectra exhibited two medium
bands at 1099, 1102 cm−1 characteristic ν(P–CPh) bands indicates the presence
of coordinated PPh3 in the complexes, similar to the literature trend [8] and sug-
gesting similarity of the structure of the two complexes. It is generally difficult
to determine the stretching frequency υ(M-P) that contains PPh3 because it has
many stretching frequencies in the lower-frequency region [9]. However, the IR
spectra of the two complexes showed interesting differences. The νCO of the three
terminal carbonyls in the tungsten complex exhibit more shift to lower frequen-
cies than that of the molybdenum complex. This can be contributed to the dif-
ference in the metal and arrangements of the ligands in the two complexes. From
the positions of the three CO groups and their intensities, it can be concluded
that the complex [Mo(CO)3(isatH)(PPh3)] could be presented in the meridional
(mer)-isomer in the solid state and tungsten complex in the facial (fac)-isomer as
shown in Scheme 1.
Table 2. Important IR data for isatin and its complexes.
Scheme 1
The 1H NMR spectrum of isatH in deuterated DMSO showed signals at

6.9(d), 7.06(t), 7.5(t), 7.61(d) ppm due to protons of the benzene ring and a
signal at 10.98(s) ppm due to proton of NH group [3]. The 1H NMR spectrum
of PPh3 showed multiplets in the range of 6.93–7.24 ppm. 1H NMR spectra
of the molybdenum complex exhibited two broad singlet signals at 11.02 and
10.86 ppm due to NH and appearance of a new doublet signal at 9.05 ppm due
to one proton in the isatin phenyl ring, in addition to the shifts of isatin and PPh3
as a result of coordination. The ratios of the signals at 11.02 and 10.86 ppm were
of (1:3) and 2:1 for molybdenum and tungsten complexes, respectively, suggest-
ing that the complex present in two-tautomeric structure. The appearance of the
new signal and change in the chemical shift of NH proton is essentially related
to the presence of the PPh3 and its effect on the chemical shifts of the isatin pro-
tons. This shift may be due to mutual anisotropic deshielding between the phenyl
group of PPh3 and one proton of the benzene ring of isatin which can affect the
signal of NH. This effect is due to magnetic field through space and not through
chemical bond by inductive effect [10]. This indicates the possibility of exchange
between CO and PPh3 groups in the solution in the axial position [11, 12] .
X-ray studies of cis-RuCl2(trpy)(PPh3); where trpy=terpyridine, showed that
the PPh3 has two phenyl rings parallel to the trpy while the third phenyl ring
nearly perpendicular to the external pyridine of trpy and this lead to low-field
shift of the parallel pyridine proton by 1.09 ppm. This was not observed for trans-
RuCl2(tepy)(PPh3) [13]. From the spectroscopic data, we can conclude that the
complexes can exist in mer- and fac-isomers in solution as shown in Scheme 2.
Scheme 2
Table 3. Absorption data of isatin and its complexes in different solvents.
Figure 1. The UV-vis spectra of the (a) [Mo(CO)3(isatH)(PPh3)], (b) [W(CO)3(isatH)(PPh3)] complexes in
different solvents.
UV-Vis Studies
The absorption spectra of isatin and its complexes were measured in ethanol.
Isatin displayed three bands at 249, 296, and 420 nm due to π-π* and n-π* transi-
tions, Table 3. The solvent effect on the position of the longer wavelength absorp-
tion band of isatin indicates that the nπ* transition has some charge transfer (CT)
character; the nitrogen atom being the electron donor and the β-carbonyl group
the acceptor. Absorption spectra of the complexes obtained from the reaction of
M(CO)6; M=Cr or Mo with isatin only as a ligand showed a shift or disappear-
ance of the CT band due to complexation through carbonyl group in isatin [3].
The electronic spectra of the complexes showed new bands in the range 360–
387 nm due to complexation and a weak band in the range of 445–490 nm. The
longer wavelength band could be attributed to metal-to-ligand charge transfer
transitions. The charge transfer bands for the [Mo(CO)3(isatH)(PPh3)] were ap-
peared at longer wavelength than the [W(CO)3(isatH)(PPh3)] Figure 1. This trend
was observed for the complexes [Mo(CO)3(pbiH)(PPh3)] and [W(CO)3(pbiH)
(PPh3)]; pbiH=2-(2′-pyridyl)benzimidazole [14].
References
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and biological activity of cycloaurated organogold(III) complexes with imidate
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acterization and biological activity of Ni, Cu and Zn complexes of isatin hydra-
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Synthesis and Characterization
of Biologically Active 10-
Membered Tetraazamacrocyclic
Complexes of Cr(III),
Mn(III), and Fe(III)
Dharam Pal Singh, Vandna Malik and Ramesh Kumar
Abstract
A new series of macrocyclic complexes of type [M(TML)X]X2; where M =
Cr(III), Mn(III), or Fe(III); TML is tetradentate macrocyclic ligand and
X = Cl−1, NO3−1, CH3COO−1 for Cr(III), Fe(III), and X = CH3COO−1
for Mn(III) has been synthesized by template condensation of succinyldihy-
drazide and glyoxal. The complexes have been formulated as [M(TML)X]
X2 due to 1:2 electrolytic natures of these complexes as shown by conductivi-
ty measurements. The complexes have been characterized with the help of ele-
mental analyses, molar conductance, electronic, infrared, far infrared spectral
studies and magnetic susceptibilities. On the basis of these studies, a five-coor-

dinate distorted square-pyramidal geometry, in which two nitrogens and two
carbonyl oxygen atoms are suitably placed for coordination toward the met-
al ion, has been proposed for all the complexes. The complexes were tested for
their in vitro antibacterial activity. Some of the complexes showed remark-
able antibacterial activities against some selected bacterial strains. The mini-
mum inhibitory concentration shown by these complexes was compared with
minimum inhibitory concentration shown by some standard antibiotics like
linezolid and cefaclor.
Introduction
During the past few decades macrocyclic chemistry has attracted the attention of
both inorganic and bioinorganic chemists. The synthesis of macrocyclic complex-
es has been a fascinating area of research and growing at a very fast pace owing to
their resemblance with naturally occurring macrocycles and analytical, industrial,
and medical applications [1–3]. In the present paper a new series of macrocyclic
complexes of Cr(III), Mn(III), and Fe(III) obtained by template condensation
reaction of succinyldihydrazide and glyoxal has been reported. These complexes
were also tested for their in vitro antibacterial activities. Some complexes showed
remarkable antibacterial activities.
Experimental
All the complexes were prepared by template method. To a stirring methanolic
solution (~50 cm3) of succinyldihydrazide (10 mmol) was added trivalent chro-
mium, manganese, and iron salt (10 mmol) dissolved in a minimum quantity of
methanol (20 cm3). The resulting solution was refluxed for 0.5 hour. After that
glyoxal (10 mmol) dissolved in ~20 mL of methanol was added to the refluxing
mixture and refluxed again for 6–8 hours. On overnight cooling, a dark colored
precipitate formed which was filtered, washed with methanol, acetone, and di-
ethyl ether and dried in vacuo (Yield 45%). The complexes were found soluble
in DMF and DMSO, but were insoluble in common organic solvents and water.
They were found thermally stable up to ~240°C and then decomposed.
Pharmacology
In Vitro Antibacterial Activity
Some of the synthesized macrocyclic complexes were tested for their in vitro an-
tibacterial activity against some bacterial strains using spot-on-lawn on Muller
Synthesis and Characterization of Biologically 305
Hinton Agar by following the reported method [4]. Four test pathogenic bacte-
rial strains viz Bacillus cereus (MTCC 1272), Salmonella typhi (MTCC 733),
Escherichia coli (MTCC 739), and Staphylococcus aureus (MTCC 1144) were
considered for determination of Minimum Inhibitory Concentration (MIC) of
selected complexes.
Culture Conditions
The test pathogens were subcultured aerobically using Brain Heart Infusion Agar
(HiMedia, Mumbai, India) at 37°C/24 hours. Working cultures were stored at
4°C in Brain Heart Infusion (BHI) broth (HiMedia, Mumbai, India), while stock
cultures were maintained at −70°C in BHI broth containing 15% (v/v) glycerol
(Qualigens, Mumbai, India). Organisms were grown overnight in 10 mL BHI
broth, centrifuged at 5000 g for 10 minutes, and the pellet was suspended in 10
mL of phosphate buffer saline (PBS, pH 7.2). Optical density at 545 nm (OD-
545) was adjusted to obtain 108 cfu/mL followed by plating serial dilution onto
plate count agar (HiMedia, Mumbai, India).
Determination of Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) is the lowest concentration of the
antimicrobial agent that prevents the development of viable growth after over-
night incubation. Antimicrobial activity of the compounds was evaluated using
spot-on-lawn on Muller Hinton Agar (MHA, HiMedia, Mumbai, India). Soft
agar was prepared by adding 0.75% agar in Muller Hinton Broth (HiMedia,
Mumbai, India). Soft agar was inoculated with 1% of 108 Cfu/mL of the test
pathogen and 10 mL was overlaid on MHA. From 1000X solution of compound
(1 mg/mL of DMSO) 1, 2, 4, 8, 16, 32, 64, and 128X solutions were prepared.
Dilutions of standard antibiotics (Linezolid and Cefaclor) were also prepared in
the same manner. 5 μL of the appropriate dilution was spotted on the soft agar
and incubated at 37°C for 24 hours. Zone of inhibition of compounds was con-
sidered after subtraction of inhibition zone of DMSO. Negative control (with no
compound) was also observed.

The analytical data show the formula of macrocyclic complexes as [M(C6H8O2N4)
X]X2. The test for anions was positive before and after decomposing the complex-
es with concentration of HNO3, indicating their presence inside as well as outside
the coordination sphere. Conductivity measurements in DMSO indicated them
to be electrolytic in nature (140–150 ohm−1 cm2 mol−1) [5]. All compounds gave
satisfactory elemental analyses results as shown in Table 1.
Table 1. Analytical data of trivalent chromium, manganese, and iron complexes derived from succinyldihydrazide
and glyoxal. Found (Calcd.) %.
IR Spectra
In the infrared spectrum of succinyldihydrazide a pair of band corresponding to
ν(NH2) is present at ~3200 cm−1 and ~3250 cm−1, but is absent in the IR spec-
tra of all the complexes. However, a single broad medium band at ~3350–3400
cm−1 was observed in the spectra of all the complexes which may be assigned due
to ν(NH). Further no strong absorption band was observed near 1710 cm−1
as observed in spectrum of glyoxal indicating the absence of >C=O groups of
glyoxal molecule. This confirms the condensation of carbonyl groups of glyoxal
and amino groups of succinyldihydrazide [6]. This fact is further supported by
appearance of a new strong absorption band in the region ~1590–1610 cm−1 in
the IR spectra of all complexes which may be attributed due to ν(C=N) [7]. These
results provide strong evidence for the formation of macrocyclic frame [8]. The
lower value of ν(C=N) indicates coordination of nitrogens of azomethine to metal
[9]. A strong peak at ~1665 cm−1 in the IR spectrum of succinyldihydrazide is as-
signed due to >C=O group of the CONH moiety. This peak gets shifted to lower
frequency (~1625–1640 cm−1) in the spectra of all the complexes [10] suggesting
the coordination of oxygen of amide group with metal.
Far Infrared Spectra

The far infrared spectra show bands in the region ~425–445 cm−1 corresponding
to ν(M–N) vibrations in all the complexes. The bands present at ~300–315 cm−1
are assigned to ν(M–Cl) vibrations. The bands present at ~220–250 cm−1 in all
nitrato complexes to ν(M–O) vibrations of nitrato group [11].
Magnetic Measurements and Electronic Spectra

Chromium Complexes
Magnetic moment of chromium complexes were found in the range of 4.0–
4.50 B.M. These values of magnetic moment support the predicted geometry of
the complexes [12]. The electronic spectra of chromium complexes show bands
at ~9030–9250, 13020–13350, 17450–18320, 27435–27840, and 34820 cm−1.
However, these spectral bands cannot be interpreted in terms of four or six coor-
dinated environment around the metal atom. In turn, the spectra are comparable
to that of five coordinated Cr(III) complexes, whose structure has been confirmed
with the help of X-ray measurements [13]. Thus keeping in view, the analytical
data and 1 : 2 ionic nature of these complexes, a five-coordinated square-pyra-
midal geometry may be assigned for these complexes. Thus, assuming the sym-
metry C4V for these complexes [14], the various spectral bands may be assigned as
4
B1→4Ea, 4B1→4B2, 4B1→4A2, and 4B1→4Eb. The complexes do not have idealized
C4V symmetry but it is being used as approximation in order to try and assign the
electronic absorption bands.
Manganese Complex
The magnetic moment of manganese complex was found to be 4.85 B.M. The
electronic spectrum of manganese complex show three d-d bands at approximately
12.250, 16.045, and 35.435 cm−1. The higher energy band at 35465 cm−1 may be
assigned due to charge transfer transitions. The spectrum resembles those report-
ed for five-coordinate square-pyramidal manganese porphyrins [14]. This idea is
further supported by the presence of the broad ligand field band at 20410 cm−1 di-
agnostic of C4V symmetry and thus the various bands may be assigned as follows:
5
B1→5A1, 5B1→5B2, and 5B1→5E, respectively. The band assignment in single elec-
tron transition may be made as d z 2 → d x2 − y 2 , d xy → d x2 − y 2 and d xy , d yz → d x2 − y 2
, respectively, in order of increasing energy. However, the complexes do not have
idealized C4V symmetry.
Iron Complexes
The magnetic moments of iron complexes lay in the range 5.82–5.90 B.M. and
are in accordance with proposed geometry of the complexes. The electronic spec-
tra of trivalent iron complexes show various bands 9825–9975, 15525–15570,
27635–27710 cm−1, and these bands do not suggest the octahedral or tetrahe-
dral geometry around the metal atom. The spectral bands are consistent with the
range of spectral bands reported for five coordinate square pyramidal iron (III)
complexes [15]. Assuming C4V symmetry for these complexes, the various bands
can be assigned as d xy → d xz , d yz and d xy → d z 2 . Any attempt to make accurate
assignment is difficult due to interactions of the metal-ligand pi-bond systems
lifting the degeneracy of the dxz and dyz pair.
Biological Assay
The minimum inhibitory concentration (MIC) shown by the complexes against
these bacterial strains was compared with MIC shown by standard antibiotics Li-
nezolid and Cefaclor (Table 2). Complex 1 showed an MIC of 8 μg/mL against
bacterial strain Escherichia coli (MTCC 739), which is equal to MIC shown by
standard antibiotic Cefaclor against the same bacterial strain. Complex 3 regis-
tered an MIC of 8 μg/mL, against bacterial strain Bacillus cereus (MTCC 1272),
which is equal to MIC shown by standard antibiotic Cefaclor against the same
bacterial strain. Further complexes 3 and 7 showed a minimum inhibitory con-
centration of 32 μg/mL against bacterial strain Salmonella typhi (MTCC 733),
which is equal to MIC shown by standard antibiotic Linezolid against the same
bacterial strain. The MIC of complex 4 against Escherichia coli (MTCC 739) was
found to be 16 μg/ml, which is equal to the MIC shown by standard antibiotic
Linezolid against the same bacterial strain. Complex 6 registered an MIC of 4 μg/
mL against bacterial strain Staphylococcus aureus (MTCC 1144) which is equal
to MIC shown by standard antibiotic Linezolid against the same bacterial strain.
Among the series under test for determination of MIC, complexes 1 and 3 were
found most potent as compared to other complexes. However, complexes 2 and 5
showed poor antibacterial activity or no activity against all bacterial strains among
the whole series. (Table 2).
Table 2. Minimum Inhibitory Concentration (MIC) shown by complexes against test bacteria by using agar
dilution assay. (—) No activity, a: Bacillus cereus (MTCC 1272); b: Staphylococcus aureus (MTCC 1144);
c: Escherichia coli (MTCC 739); d: Salmonella typhi (MTCC 733); Cefaclor and Linezolid are standard
antibiotics.
Conclusions
Chemistry
Based on elemental analyses, conductivity and magnetic measurements, elec-
tronic IR, and far IR spectral studies, the structure as shown in Figure 1 may be
proposed for these complexes.
Figure 1
Biological Assay
It has been suggested that chelation/coordination reduces the polarity of the metal
ion mainly because of partial sharing of its positive charge with donor group
within the whole chelate ring system [16]. This process of chelation thus increases
the lipophilic nature of the central metal atom, which in turn, favors its perme-
ation through the lipoid layer of the membrane thus causing the metal complex
to cross the bacterial membrane more effectively thus increasing the activity of
the complexes.
Abbreviations
MIC: Minimum inhibitory concentration
MTCC: Microbial type culture collection
MHA: Muller Hinton Agar
CFU: Colony forming unit

B.M.: Bohr Magneton
DMF: N,N-dimethylformamide
DMSO: Dimethylsulphoxide
BHI: Brain heart infusion
Acknowledgements
D. P. Singh thanks the University Grants Commission, New Delhi for financial
support in the form of Major Research Project. Thanks are also due to authori-
ties of N.I.T., Kurukshetra for providing necessary research facilities. The authors
are thankful to Dr. Jitender Singh for carrying out the biological activity of the
synthesized macrocyclic complexes.
References
1. K. Gloe, Ed., Current Trends and Future Perspectives, K. Gloe, Ed., Springer,
New York, NY, USA, 2005.
2. L. F. Lindoy, Ed., The Chemistry of Macrocyclic Ligand Complexes, L. F.
Lindoy, Ed., Cambridge University Press, Cambridge, UK, 1989.
3. E. C. Constable, Ed., Coordination Chemistry of Macrocyclic Compounds,
E. C. Constable, Ed., Oxford University Press, Oxford, UK, 1999.
4. D. P. Singh, R. Kumar, and J. Singh, “Synthesis and spectroscopic studies of
biologically active compounds derived from oxalyldihydrazide and benzil, and
their Cr(III), Fe(III) and Mn(III) complexes,” European Journal of Medicinal
Chemistry, vol. 44, pp. 1731–1736, 2009.
5. R. Kumar and R. Singh, “Chromium(III) complexes with different chromo-
spheres macrocyclic ligand, synthesis and spectroscopic studies,” Turkish Journal
of Chemistry, vol. 30, no. 1, pp. 77–87, 2006.
6. Q. Zeng, J. Sun, S. Gou, K. Zhou, J. Fang, and H. Chen, “Synthesis and spec-
troscopic studies of dinuclear copper(II) complexes with new pendant-armed
macrocyclic ligands,” Transition Metal Chemistry, vol. 23, no. 4, pp. 371–373,
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7. L. K. Gupta and S. Chandra, “Physicochemical and biological characterization
of transition metal complexes with a nitrogen donor tetra-dentate novel macro-
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synthesis of 14–16 membered tetraimine macrocyclic complexes,” Transition
Metal Chemistry, vol. 24, no. 2, pp. 198–201, 1999.
9. C. Lodeiro, R. Bastida, E. Bértolo, A. Macías, and A. Rodríguez, “Synthesis and
characterisation of four novel NxOy-Schiff-base macrocyclic ligands and their
metal complexes,” Transition Metal Chemistry, vol. 28, no. 4, pp. 388–394,
2003.
10. D. L. Pavia, G. M. Lampman, and G. S. Kriz, Introduction to Spectroscopy,
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11. M. Shakir, K. S. Islam, A. K. Mohamed, M. Shagufta, and S. S. Hasan, “Mac-
rocyclic complexes of transition metals with divalent polyaza units,” Transition
Metal Chemistry, vol. 24, no. 5, pp. 577–580, 1999.
12. D. P. Singh and R. Kumar, “Trivalent metal ion directed synthesis and charac-
terization of macrocyclic complexes,” Journal of the Serbian Chemical Society,
vol. 72, no. 11, pp. 1069–1074, 2007.
13. J. S. Wood, “Stereochemical electronic structural aspects of five-coordination,”
Progress in Inorganic Chemistry, vol. 16, p. 227, 1972.
14. D. P. Singh and V. B. Rana, “Binuclear chromium(III), manganese(III), iron(III)
and cobalt(III) complexes bridged by diaminopyridine,” Polyhedron, vol. 14,
no. 20-21, pp. 2901–2906, 1995.
15. A. B. P. Lever, Inorganic Electronic Spectroscopy, Elsevier, Amsterdam, The
Netherlands, 1984.
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Antifungal and Spectral
Studies of Cr(III) and Mn(II)
Complexes Derived from
3,3′-Thiodipropionic Acid
Derivative
Sulekh Chandra and Amit Kumar Sharma
Abstract
The Cr(III) and Mn(II) complexes with a ligand derived from 3,3′-
thiodipropionic acid have been synthesized and characterized by elemental
analysis, molar conductance measurements, magnetic susceptibility measure-
ments, IR, UV, and EPR spectral studies. The complexes are found to have
[Cr(L)X]X2 and [Mn(L)X]X, compositions, where L = quinquedentate li-
gand and X=NO3−, Cl− and OAc−. The complexes possess the six coordinated
octahedral geometry with monomeric compositions. The evaluated bonding
Antifungal and Spectral Studies of Cr(III) and Mn(II) 313
parameters, Aiso and β, account for the covalent type metal-ligand bonding.
The fungicidal activity of the compounds was evaluated in vitro by employ-
ing Food Poison Technique.
Introduction
The synthesis of the coordination compounds of the Schiff’s base ligands having
N,S-donor binding sites has attracted a considerable attention because of their po-
tential biological activities [1–3]. The main features of these compounds are their
preparative accessibility, diversity, structural variability and versatile coordinating
properties. These compounds have also been widely investigated to examine the
effect of metallation on the antipathogenic activities of such ligand systems. The
studies of antipathogenic behavior of these chemically modified species are of
paramount importance for designing the metal-based drugs. These compounds
have been found to be more effective when they are administered as metal com-
plexes [4–6].
In view of these aspects and our preceding work, we report here the synthesis,
spectral, and antifungal studies of Cr(III) and Mn(II) complexes derived from
ligand, 3,3′-thiodipropionic acid bis(4-amino-5-ethylimino-2,3-dimethyl-1-phe-
nyl-3-pyrazoline).
Experimental
The ligand 3,3′-thiodipropionic acid bis(4-amino-5-ethylimino-2,3-dimethyl-
1-phenyl-3-pyrazoline) (Figure 1) was synthesized according to the literature
method [7]. The complexes were synthesized by refluxing 1 mmol of the metal
salt (nitrate, chloride, and acetate) with 1 mmol of ligand in acetonitrile for 8–14
hours at 70–80°C. The resulting mixture was kept in refrigerator overnight at
0°C. The solid powder was filtered, washed with cold acetonitrile and dried under
vacuum over P4O10.
Figure 1. Structure of ligand.

The fungicidal activity of the compounds was screened in vitro by employing

Food Poison Technique [7] against the plant pathogens viz. Alternaria brassicae,
Aspergillus niger, and Fusarium oxysporum.
Microanalytical analyses were performed on a Carlo-Erba 1106 analyzer. IR
spectra were recorded as KBr pellets in the region 4000–200 cm-1 on an FT-
IR spectrum BX-II spectrophotometer. The electronic spectra were recorded on
Shimadzu UV mini-1240 spectrophotometer using DMSO/DMF as a solvent.
EPR spectra were recorded in solid and solution forms on an E4-EPR spectrom-
eter at room temperature and liquid nitrogen temperature operating in X-band
region. The molar conductance of complexes was measured in DMSO/DMF at
room temperature on an ELICO (CM 82T) conductivity bridge. The magnet-
ic susceptibility was measured at room temperature on a Gouy balance using
CuSO4.5H2O as callibrant.

The microanalytical data, magnetic moments, and other physical properties of
complexes are summarized in Table 1. As we reported earlier [7], the ligand co-
ordinates to the metal atom in the NNSNN fashion via five binding sites and
forms the stable complexes having [Cr(L)X]X2 and [Mn(L)X]X compositions.
The molar conductance value accounts for the 1:2 and 1:1 electrolytic nature of
Cr(III) and Mn(II) complexes, respectively, (Table 1) [8]. The magnetic moments
of these complexes lie in the range 3.78–3.89 (CrIII) and 5.89–5.98 B.M. (MnII).
Table 1. Analytical data, magnetic moments, and physical properties of complexes.
The IR spectrum of the free ligand shows bands at 1647, 1621, 1532, 768
cm-1 due to ν(C=O) amide I, ν(C=N) azomethine, NH in-plane-bending (amide
III) vibrations and ν(C–S), respectively. On coordination, the position of ν(C=N),

amide III and ν(C–S), bands is altered, which indicates that the nitrogen atoms of
C=N and NH groups, and the sulphur atom of the C–S group are coordinated to
the central metal atom. Further, the IR spectrum of the ligand also shows a band
at 3225 cm-1 due to the ν(NH) stretching vibration. On coordination, this band
shows a negative shift, which is in further support of coordination of the NH
group through nitrogen. However, the amide I band does not show any consider-
able change in its position on complexation, which suggests that the C=O group
does not participate in coordination [7, 9, 10]. The IR spectra of complexes also
give the new bands at 407–497 and 312–328 cm-1 due to ν(M–N) and ν(M–S)
stretching vibrations [7, 11]. This discussion reveals that the ligand coordinates to
metal atom in the NNSNN manner. The complexes also show the IR bands due
to coordinated anions [12].
The electronic spectra of complexes were recorded in DMF/DMSO solution.
The electronic spectra of Cr(III) complexes exhibit the absorption bands in the
range 13280–19231, 25028–27027, and 36764–37735 cm-1 due to the 4A2g →
4T2g(F)(ν1), 4A2g → 4T1g(F)(ν2), and 4A2g → 4T1g (P)(ν3) spin allowed d-d
transitions, respectively. These bands suggest an octahedral geometry for Cr(III)
complexes (Figure 2) [13].
Figure 2. Structure of [Cr(L)X]X2 complexes, where X = NO3-, Cl- and OAc-.
The electronic spectra of Mn(II) complexes show the absorption bands in the
range 16970–19540, 22280–24390, and 26109–27624 cm-1. These absorption
bands may be assigned to the 6A1g → 4A1g (4G), 6A1g → 4A2g(4G), and 6A1g
→ 4Eg, 4A1g (4G) transitions, respectively. These bands suggest that the com-
plexes possess an octahedral geometry [13]. The complexes also show the band
in the region 34843–38022 cm-1 due to a charge transfer transition. Different
ligand field parameters have been evaluated for the complexes and the value of
covalency factor β (0.43–0.79) reflects the covalent nature of the L → M bond.

The covalency factor β was evaluated by using the expression β=Bcomplex/Bfree
ion, where B is the Racah interelectronic repulsion parameter. The value of B
lies in the range 542–784 and 418–763 cm-1 for Cr(III) and Mn(II) complexes,
respectively.
The X-band EPR spectra for Cr(III) complexes in solid form show a broad
signal at giso= 1.9829–2.2870. The signal does not show hyperfine splitting due
large line widths. The EPR results of Cr(III) complexes are consistent with the
presence of hexacoordinated Cr(III) centers [14].
The EPR spectra for Mn(II) complexes in solid form give broad signal at giso=
1.9763–2.1351 both at room temperature and at liquid nitrogen temperature.
However, the EPR spectra of complexes in solution (RT and LNT) show the
hyperfine splitting and give six lines at giso= 1.9835–2.5961 (55Mn, I=5/2). The
hyperfine coupling constant Aiso was evaluated and its values (90.0–96.0) are
consistent with the complexes having Mn(II) central metal atom in an octahedral
field [15].
The results of the antipathogenic activity of compounds are summarized in
Table 2. The fungal inhibition capacity of the compounds was compared with the
standard fungicide Captan. The data indicate that the complexes possess greater
fungicidal activity in comparison to ligand which is due to their higher lipophilic-
ity. This modified fungicidal behaviour of the complexes is based on the Over-
tone’s Concept and Chelation Theory [7].
Table 2. Antifungal activity data of the compounds.
Conclusions
The spectral analysis of the compounds reveals that the ligand acts as quinque-
dentate chelate and bound to the metal atoms through NNSNN-donor sites.
The bonding parameters account for the covalent nature of L → M bond. The
complexes are six coordinated with metal atom surrounded by an octahedral co-
ordinating species. The screening of fungicidal activity of compounds led to the
conclusion that complexes possess moderate antipathogenic behavior than the
free ligand.
Acknowledgements
The authors sincerely express their thanks to DRDO, New Delhi financial sup-
port and Dr. P. Sharma, Principal Scientist, IARI, Pusa, New Delhi for providing
laboratory facility for determining the fungicidal activity.
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Copyrights
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ed use, distribution, and reproduction in any medium, provided the original
author and source are credited.
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tribution 3.0 License.
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under the Creative Commons Attribution License, which permits unrestricted
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17. Copyright © 2009 Dharam Pal Singh et al. This is an open access article dis-
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Trimm
Inorganic Chemistry
Reactions, Structure and Mechanisms Research Progress in Chemistry
Inorganic chemistry is the study of all chemical compounds except those containing carbon, which
is the field of organic chemistry. There is some overlap since both inorganic and organic chemists
traditionally study organometallic compounds. Inorganic chemistry has very important
ramifications for industry. Current research interests in inorganic chemistry include the discovery
Inorganic Chemistry
of new catalysts, superconductors, and drugs to combat disease. This new volume covers a
diverse collection of topics in the field, including new methods to detect unlabeled particles, Reactions, Structure and Mechanisms
measurement studies, and more.
Inorganic Chemistry
About the Editor
Dr. Harold H. Trimm was born in 1955 in Brooklyn, New York. Dr. Trimm is the chairman of the
Chemistry Department at Broome Community College in Binghamton, New York. In addition, he is
an Adjunct Analytical Professor, Binghamton University, State University of New York,
Binghamton, New York.
He received his PhD in chemistry, with a minor in biology, from Clarkson University in 1981 for his
Harold H. Trimm, PhD
work on fast reaction kinetics of biologically important molecules. He then went on to Brunel Editor
University in England for a postdoctoral research fellowship in biophysics, where he studied the
molecules involved with arthritis by electroptics. He recently authored a textbook on forensic
science titled Forensics the Easy Way (2005).
Other Titles in the Series

• Analytical Chemistry: Methods and Applications
• Organic Chemistry: Structure and Mechanisms
• Physical Chemistry: Chemical Kinetics and Reaction Mechanisms
Related Titles of Interest

• Environmental Chemistry: New Techniques and Data
• Industrial Chemistry: New Applications, Processes and Systems
• Recent Advances in Biochemistry
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Harold H. Trimm, PhD, RSO

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International Standard Book Number-13: 978-1-4665-5977-6 (eBook - PDF)

6. Development of Inorganic Membranes for Hydrogen Separation 173

15. Kinetic and Mechanistic Studies on the Reaction of 286

— Harold H. Trimm, PhD, RSO

Eleonora Zakharian, Baskaran Thyagarajan, Robert J. French,

biochemical analysis of the TRPM8 protein also uncovered the presence of

Intracellular Ca2+ Measurements

Preparation of the TRPM8 Protein

concentrated to a final concentration of 12 µg/ml and analyzed by Western blot

Planar Lipid Bilayer Measurements

between symmetric aqueous bathing solutions of 150 mM KCl, 20 mM Hepes,

possible effect of polyP on TRPM8 we conducted a number of experiments with

We next tested the effect of scPPX1 by transiently transfecting scPPX1-pcD-

expressing GFP alone. In controls, the values of menthol-induced currents ob-

In order to test whether the significant inhibition of TRPM8 channel activity

Inorganic Polyphosphate and Polyhydroxybutyrate Associate

The presence of polyP in tetramers of TRPM8 was detected by its metachro-

Association of polyP with proteins has frequently been found to be mediated

Inhibition of TRPM8 Channel Activity by scPPX1 in Planar

aqueous solutions of 150 mM KCl, 0.2 mM MgCl2 in 20 mM Hepes, pH 7.2.

All the following bilayer experiments were conducted in presence of 1%

Figure 6. A. Representative current/voltage relationship of TRPM8: Channels were incorporated in planar

Further, we examined the effect of scPPX1 on the single channel activity of

Figure 9. Reduction of TRPM8 channel conductance by exopolyphosphatase scPPX1. A: Representative single-

Next we analyzed the effect of scPPX1 on channel conductance. We found

Inorganic polyphosphate is present in all eukaryotic cells, however its func-

evidence that PtdIns(4,5)P2 regulates these channels through direct association,

rapid, incompletely resolved gating transitions, or a “fast block” process, due to

11. Liu B, Qin F (2005) Functional Control of Cold- and Menthol-Sensitive

25. Kornberg A, Rao NN, Ault-Riche D (1999) INORGANIC POLYPHOS-

TOL (4,5)-BISPHOSPHATE (PI(4,5)P2) IS THE ENDOGENOUS LIPID

48. Zakharian E, Reusch RN (2004) Functional evidence for a supramolecular

dramatically reduced by the simultaneous consideration of various bioener-

Energetic, Physical, and Geological Constraints on

Physics: Photostability of Nucleotides

after a UV-irradiation, the self-cleavage activity of the montmorillonite-adsorbed

Geology: Requirement for Hydrothermal Settings

The above listed constraints, when considered simultaneously, identify crystal-

Hypothesis: Emergence of the First Biopolymers

Figure 1. Primeval ZnS-mediated photosynthesis in sub-aerial, illuminated settings. Right: Precipitation of

First Settlers in the ZnS World

First Replicating Entities

1) Nucleobases quench the UV quanta by converting the energy into heat.

Emergence of Proteins and Enzymes

The First Colonization Wave

The Fall of the Zn World

Validation of the Hypothesis

Initial Geological Settings

In summary, these features point to environments similar to those in which

Photosynthesis in the Zn World

was prevented by the reductive action of hypophosphite that yielded phosphite,

s22− + H 2 PO2− + 3OH − → 2 S 2− + HPO3− + 2 H 2O (3)

S 22− + HPO3− + 3OH − → 2 S 2− + HPO43− + 2 H 2O (4)

The high efficiency of (hypo)phosphite as a hole scavenger was also demon-

First Settlers in the ZnS World

It has been argued that biological stereoselectivity (homochirality), i.e. the