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Heparan sulphate microstructure, evidently strictly regulated both temporarily and positionally during
development (being responsible amongst numerous other established functions for regulating several
growth factor activities [(1) cf Bernfield et al 1999; Hacker et al 2005] which are apparently dependent
on the existence in syndecans, glypicans, agrin and other protoglycans of heparan sulphate sidechains
having a great variety different linearly information-encoded sequences (dependent on the occurrence
in this uronic acid (1->4) D glucosamine repeat disaccharides of sulphated iduronate, glucouronate and
N-acetyl, N-sulphonate or unsubstituted glucosamine 2-deoxyglucosamine, residues) which are
believed to enable several types of signalling including those involving binding to basic sites in
proteins via specific information encoded sequences of sugars analogous to the pentasaccharide
antithrombin-binding site heparin [(1) Bernfield et al 1999; Lyon & Gallagher 1997] (such an
information code is more complex than that of DNA to which it is obviously is however similar in
principle although the heparan sulphate system apparently lacks any ability to act as a template for its
self replication).
It is now further suggested that binding of polysaccharides to proteins may require correct interstitial
aqueous solutions (e.g. containing separate ‘soft-ice’ like phases) which are now believed to contribute
to the inorganic co-solute multi-ionic environments** .
With heparan sulphate it seems that a system other than molecular recognition by an antithrombin
(AT)-like-fine tuned electrostatic quaternary N+ protein link to individual codon sulphate mineral-like
a
anionic patterns (cf ) must allow fine structure discrimination to be established. A required high
degree of selectivity by different microstructures present in different heparan sulphates seems to be the
basis for their ability to select different proteins for their normal (e.g. growth factor orchestration)
functions (1). The existence of some previously unsuspected unconventional molecular recognition
system is however apparently required to facilitate this. This possibility is suggested by the work of
[(1a) Kreuger et al 2005] who found that most probable signalling oligosaccharides failed to
discriminate between their individual FGF growth factor isoform target binding sites when they were
present as individual molecular signals (obtained from the high molecular weight by scission,
separation and purification).
This finding seems to be starkly contrary to the selectivity apparently achieved by the parent high
molecular weigh proteoglycans in their in vivo environments.
This situation might have arisen (as suggested by Kreuger et al) from the ability of such larger
molecules (but not the smaller segments) to form additional hydrogen-bonding, Van der Waals forces
and salt links (including those with inorganic ion bridges [cf 1a-1]) and perhaps involving the whole
heparan sulphate proteoglycan chain which may be involved.
It is now proposed that the ultimate basis of heparan sulphate signalling is actually the induced water
structure (nb this is commonly acknowledged to be promoted in some non-specific manner by the
extracellular matrix) and that this is the ultimate cause of tissue integrity; tissue development may also
require input form ‘correct’ water structures which might be associated with the longer polysaccharide
segments molecules. This correct water structure for the proposed heparan sulphate signalling etc.
functions could involve both hydrophilic and hydrophobic repulsive and attractive forces similar to the
often long range effects which have been identified to occur in aqueous solutions adjacent to
hydrophobic surfaces and hydrophobic and hydophilic mica surfaces (cf. [(1b) Christenson & Claesson
2001] for which puzzling long range effects have been identified which might conceivably mimic the
high anionic density + hydrophobic N-Ac region systems of heparan sulphates). These types of water
structures are also believed to critically depend on the presence of sub-microscopic sized nano holes
which have been generated in water structures by the micro-bubbles (which exist adjacent to mica and
silicate surface studied by atomic force microscopy) pointing to the possible key role of such a
mechanism in the of formation of (presumable non-equilibrium, thermostatically unstable) water
microstructures in the biochemical mechanism of tissue generation and its upkeep.
Myelin basic protein integrity may depend on such hydrophobic induced water structure (and depend
on micro-bubbles for its existence) as it is strongly associated with lipid induced hydrophobic waters
structuring effects [(1c) Muelle et al 1999)] . It should be noted that the integrity of this protein has
also been associated with heparan sulphate proteoglycans which enable the repair of damaged myelin
sheaths, defects in which can be argued to give rise to neurological conditions such as multiple
sclerosis. A scenario by which is this process becomes disrupted by a UV-vitamin D-thyroid factor
dependent sulphate transporter which facilitates heparan sulphate sulphation could explain the
geographical incidence of multiple sclerosis in Australia and perhaps also the possible promotion of
this disease by barium intoxication [(1d) Purdey, 2004].
Biological Fluids are Seawater-Like Multielement Matrices from which Heparin and Anionic
Polysaccharides Selectively Sequester the Least Abundant Solute Ions.
In a internet paper (now no longer accessible?) discussing how water structure influences protein
folding G Wilse Robinson quoted
Szengt Gorgi who stated that humans could be described as bags of skin filled with seawater.
That seawater differs markedly from pure water points to the importance of such differences for
biochemistry and the need to centre biochemical studies on a more detailed model of impure water
(especially the effect of colloids in seawater and associated dissolved gases which might impart
nanobubble structures and confer long range water structuring effects).
The multi-inorganic nature of geological and biological phenomena including the seawater range of
inorganic elements.
The overlap of geology, inorganic chemistry and biological chemistry suggested a new branch of
science was required (“metallomics”).
This might , it can be argued rationally centre around the effect of water structure.
[(3) Haraguchi, 2004] included heparin (but apparently only in regard to its sulphur content) in his
tabulation of topics for which a suggested new science [dealing with the] should be potentially
considered of fundamental importance to biochemistry since biological fluids were multi-inorganic ion
solutions which were approximately similar to seawater and other natural waters.]
It might further be suggested that since heparan sulphate seems to have co-evolved with multicellualr
animals in the sea some 109 years ago a primitive role of cell surface heparan sulphates was to act as a
nutrient gatherer and buffer for seawater-like multi inorganic element containing salt solutions.
This notion seems to be confirmed by a report from the Dietrich group of the existence of an exact
mathematical relationship [(4) Nader et al. 1983) between the amounts of tissue heparan sulphates (and
other sulphated polysaccharides) and the salinities of the habitats of fifteen species of aquatic
invertebrates, where habitat water might be required to directly bathe the heparan -sulphate-
proteoglycan-lined tissues.
A more evolved system in vertebrates which use complex hormone and dedicated kidney water ion
homeostasis is that these organs seem to have the ability to respond to the presence of small ions which
might adversely affect their tissues by up-regulating heparan sulphate anti-crystal activity (an activity
which is probably dependent on water structure modulation) [kidney epithelial cells have been reported
to alter their incorporation of radiolabelled sulphate into heparan sulphate and chondroitin sulphate in
response to oxalate as well aas Ca oxalate crystals which are inhibitable by these polysaccharides [(4a)
Borges et al 2005]].
Anionic polysaccharides (and proteins) in animals when bathed in the multi-inorganic ion salt solution
biological fluids will generate multi-inorganic ion/hydration water complexes similar to the anionic
polysaccharides abundantly present in the cell walls of marine algae had been established [(4b)
Wassemann, 1949] most likely to exist in this form in vivo rather than being present, as was originally
supposed, as free alginic acid.
Although less chemically definable than the pure polyanionc polysaccharides (alginates, carrageenans,
and the polysaccharide side-chains of glycosaminoglycans etc.) but nevertheless of considerable
importance to the homoeostasis of inorganic ions including carbonate, bicarbonate and Ca2+ ions in
natural waters is the system of humic/fulvic polymers (a system of polymethylene, polycarbonyl,
caboxylated) material which comprises the largest system of organic polymers on earth. [Added later,
cf e.g. DA Hansell et al “Dissolved Organic Matter in The Ocean”, Oceanography 2009 22(4) “at
662PgC marine dissolved organic matter contains as much carbon as the atmosphere and is one of the
Earth’s major carbon reservoirs”] These natural polyanions bind numerous metal ions present in
seawater etc. via abundant -COO- groups, which apparently gave rise to the geological deposits of
fulvate organic matter.
Determination by spark source mass spectrometry (SSMS) of the multi-inorganic element contents of
geological fulvates and marine alginate showed obvious qualitative similarities to the SSMS results
for the multi-element contents of the animal polysaccharide heparin [(5) Grant et al 1987].
Less information is currently available from the literature of similar studies of heparan sulphates
(which are more difficult to obtain in large amounts) but studies conducted in the context of
scitigraphic imaging (e.g. of tumours) has indicated that this procedure may depend upon the binding
of the radio-nuclides to heparan sulphate proteoglycans e.g. at cell surfaces; side experiments
established that 45Ca in heparan sulphate could be replaced by a range of multivalent metal counterions
in a manner consistent with heparan sulphate being normally present in vivo in the form of a multi-
inorganic matrix (5a).
The apparent differences in the observed biochemical/physical properties of different brands of heparin
seems to at least in part have its origin in the different degrees of ‘purification’ achieved by different
manufacturers (6). It might even be suggested that such attempts at purification actually achieve
inappropriate forms of heparin, at least from the requirement of biochemical if not from the standpoint
of pharmacological research.
What is obviously required for fundamental biochemical researches is the actual form of polyanions
which are present in vivo. That the achievement of the equilibrium between the polyanion and the
multi-element bathing fluids is not re-established rapidly may be deduced form the reports that the
different single salt forms of heparin have different in vivo activities.
The traditional view was that the multi-element character of unrefined heparin was of little scientific
interest and that samples used for biochemical researches should be as free from such multi-elements as
possible. [A similar hypothesis was applied to chitosan research, where the existence of multi-
elements had been attributed to uptake from a final washing in tap water].
This idea is suggested to be incorrect and the status of the inorganic/water co-sphere around anionic
polysaccharides which could potentially be part of an organometallic signallling system needs to be re-
evaluated.
*This hypothesis was generated from private discussions (including with FB Williamson PhD and
Professor WF Long** of the former University of Aberdeen, Marishal College Polysaccharide
Laboratory, correspondence with Professor RJP Williams , Oxford University) and extensive internet
literature studies made privately from Ashbank, Turriff AB53 6SX, UK as well as by use of the
facilities kindly provided by Queen Mother Library King’s College and Forresterhill Medical Library,
University of Aberdeen.
**The majority of the published papers of this polysaccharide research group are given in
http://www/abdn.ac.uk/~bch118/publicatuions2003march.doc
a
( ) Grant D Tait MI Long WF Williamson FB
Microstructure-dependent crystallisation modulation by alginates
Grant D Somers JA Tait MI Long WF Williamson FB
Anti-calcite crystallisation activities of carrageenans
Posters presented by MI Tait at the XIIIth International Seaweed Symposium Vancouver 13-18
August, 1989
b
( ) Protein folding was traditionally viewed as an intrinsic property of the amino acid sequence in
which the solvent had a secondary role; inherent hydrophilic/hydrophobic effects were believed to be
directed by the amino acid sequences alone. This view has recently been challenged.
A recent re-evaluation of how proteins fold now suggests that the principal influence (or driving force)
for protein folding originates from a dominant effect of water structure/activity on protein
supramolecular structure/conformation.
Such an influence of water structure also explains the Hofmeister or lyotropic series a phenomenon of
protein denaturation characteristic of high solute (including inorganic salt) molecule concentrations.
Cf Kleeberg H 1987
Proc Symposium in Honor of WAP Luck Marburg FRG
Sasisekharan R et al 2002
Nature Rev Cancer 2(7) 521-528;
Park PW et al 2000
J Biol Chem 275 29923-29926
Cf also Esko JD Lindahl U. Suggested reading list entitled ” Molecular diversity of heparan sulfate”
available at
http://www.jci.org/content/full/108/2/169/DC1
Added later
(1a-1) Rudd TR et al 2007, 2008, 2009
Glycobiology 17 (9) 983-993; ibid., 19 (1) 52-57
Carbohydr Res 343 2184-2193
Israelachvile J 1987
PNAS USA 84 4722-4724
3) Haraguch H (2004)
J Anal At Spectrom. 19, 5-14
4) Nader HB Medeiros MGL Paiva JF Paiva VMP Jeronimo SMB Ferreira TMPC Dietrich CP
1983
Relationship between habitat salinity and the average total
sulphated glycsoaminoglycan contents in fifteen species of Crustacea, Pelecypoda and Gastropoda
Comp Biochem Physiol. 76, 433-436
The original report of these SSMS data was made by Moffat CF Ph D Thesis
Synthesis, Characterizsation and Applications of Chemically Modified Heparins
University of Aberdeen 1987 p 187-189
6)
Added later:
web.scribd.com/doc/26994439/Publication-2-Web
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Second Manuscript
The yet-to-be-achieved rationalisation of the compensation effect could point to the how ultimate
basis of water structure might be elucidated
N.b. strict entropy enthalpy compensation is also found for many systems of related rate constants in
organic chemistry (cf. Leffler JE 1955 loc cit).
The origin of the effect, however, is at present not established.
It seems likely that some rigorous mathematical description might be possible which might predict the
outcome of processes which are considered at present to be governed by the usual thermodynamic
principles; this may especially apply to pseudo equilibria where different pathways are used for the
forward and reverse reactions and are therefore such processes are not fully reversible (this situation
might apply to the supramolecular rearrangements which occur in water rich polysaccharide gels which
demonstrate a pronounced hysteresis of melting and the apparent occurrence of water ‘memory’ cf.
Martin Chaplin 2008 “Water Structure And Science” internet http://www.Isbu.ac.uk/water/ref5.html
[this includes a list of references which might support the existence of a water memory effect e.g. ref.
500 (L Rey, 2004) suggests that simple salt solutions when diluted beyond the Avogadro number retain
memory of the alklai metal salt); the “Memory of Water” article however also concludes that the
apparent memory effect of water is probably due to solute and surface changes occurring during
processing (for which homeopathic usage prefers glass rather than polypropylene vessels, both of these
can inject ultratrace amounts of inorganic Al2O3 and TiO2 particles which like SiO2 can in principle
create very high water content gels). M Chaplin believed that the main evidence against the existence
of water memory, per se, however, was that hydrogen bonded structures would be required reform their
structures to allow this (despite the short. viz. picosecond lifetimes for the hydrogen bonds and
milliseconds for H-O bonds in liquid water such lifetimes would not necessarily control the lifetimes of
clusters which were suggested to be able to simultaneously disintegrate and reform identical
structures).
If water memory is real then water structure exchanges cannot be held under thermodynamic reversible
equilibrium conditions which would require
all water-only-phases to eventually produce identical final distributions of aggregates and clusters
The existence of order in chaotic systems however could indicate that stable configurations even of
unstable linkages undergoing interchanges in liquid water (especially the Si etc. multi-element
seawater-like aqueous solution which can be argued (cf Haraguchi 2004 J Anal At Spectrom 19 5-14)
to be the correct form for biochemical studies) could permit memory effects to persist for longer
periods of time. This requires a chemical engineering fluid dynamic approach to water structure and
the influence of true seawater or blood serum including correct amounts of the 50+ trace and ultratrace
elements).
The enthalpy-entropy “compensation effect” had usually been dismissed as an artifact or experimental
error since it was apparently not exhibited, even approximately, by many (or most?) chemical
reactions.
However dismissal of the compensation effect is felt to be unjustified when the full range of reported
instance of this effect is taken into account (cf. Boon, MR 1973 Nature 243 401 and following letters in
the journal). It is applicable, as shown by Barclay & Butler in 1938 (Trans Faraday Soc. 34, 1445-
1554) to the heats and entropies of vaporisation from solutions and pure un-associated liquids and, as
shown by Campbell & Eley in 1940 (Trans Faraday Soc 36, 854-856) and Eley in 1967 (J Polymer Sci
C (17) 73-91 to the electrical conductivity of solids (including amino acids and proteins) (possibly
influenced by strongly held water), to the electrical conductivity of organic and inorganic oxides
(including TiO2, Fe2O3 and ZnO) to the thermionic emission of electrons from solid surfaces and the
transfer of ions and molecules between H2O and D2O. It also applies to the haemolysis of erythrocytes
and to the denaturation of proteins (Brown HD Ed 1971, in Chemistry of the Cell Interface, Part B,
Academic Press New York, cf. p 70) as well as to many heterogeneous catalytic processes including
hydrocarbon formation on clays (Wilson MC. & Galwey AK 1973, Nature 243, 402-404.
It applies to nucleated disorder-order transition in kappa carrageenan (which was reported but not
discussed by Austin KRJ et al in 1988 (Biopolymers 27, 139-155) the results presented by these
authors suggest an isokinetic temperature around 20C); such nucleation rates also follow the
Hofmeister (lyotropic) series which was correlated with water aggregation by WAP Luck (as discussed
in Kleeberg H (Ed) by multiple authors 1987, in the Proceedings of a symposium in honour of WAP
Luck on interactions of water in ionic and non-ionic hydrates held in Marburg FRG published by
Springer Verlag).
Hints as to how further insight and rationalisation of compensated system behaviour might be achieved
might be gleaned from systems where randomisation of chemical structure interchanges occurs {a
review of such phenomena in inorganic chemistry is given by Van Wazer JR 1962 Amer Sci 50, 450-
472}; a useful model of water structure could perhaps also be developed according to the
randomisation of structures similar to those discussed here (later work by Van Wazer et al. also
established that, in addition to the occurrence of general randomisation in many systems of e.g.
inorganic polymers, ring and cluster formation was also of especial importance); since Van Wazer et
al. believed that these phenomena could be accommodated within traditional thermodynamic
principles, it is now however suggested that a different mathematical treatment is required.
Further experimental work by the author on one of the systems where structural reorganization had
been established to occur (chlorocarbon scrambling under sealed tube pyrolysis conditions; Grant D
(1974 loc cit); also to be the subject of a further discussion article) had suggested that the assumption
of attainment of a full thermodynamic reversibility in this system is incorrect.
***A, widely applicable throughout nature, general entropy and enthaply compensation system also
applies to the surface tensions of liquids which can be considered to be limited to values which obey
this relationship {Campbell H Eley DD 1940 loc cit}; this will putatively also govern the allowable
surface tension changes associated with the Hofmeister effect which is dependent on water structure
alteration.