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Cell Biology International, 1996, Vol. 20, No.

6, 429435

HIGH AND LOW DENSITY WATER AND RESTING, ACTIVE AND


TRANSFORMED CELLS

PHILIPPA M. WIGGINS

Department of Medicine, University of Auckland Faculty of Medicine and Health Science, Private Bag 92019,
Auckland, New Zealand

Accepted 22 April 1996

Resting and active states of cells are described in terms of the expectation, derived from
experiments with aqueous polymers, that they contain two modified forms of water: high
density, reactive, fluid water and low density, inert, viscous water. Low density water
predominates in a resting cell and is converted to high density water in an active cell. It is
proposed that switching from one state to another is an integral part of cellular function.
When this ability is lost cells are transformed either to a state of rigor or to a hyperactive
state in which they no longer depend upon external signals. ? 1996 Academic Press Limited

K: water; cytomatrix; osmosis.

INTRODUCTION High and low density water

Highly charged polymeric solutions and gels have These states of water are metastable and transient
been shown to contain two modified forms of but they appear to last long enough to play sig-
water, each of which differs in all measured nificant roles in biochemical and physiological
properties from normal water (Wiggins, 1994a,b, processes. Their formation is determined by both
1995a,b,c). Low density water is more strongly thermodynamic and kinetic considerations. In
hydrogen-bonded, more viscous and, relative to simple solutions a gradient in osmolality is abol-
normal water, is highly selective in its solvent ished by movement of water and solutes until their
properties. High density water is more fluid than chemical potentials are equal throughout. That is
normal water and, again, relative to normal water the state of lowest free energy. In aqueous polymer
and to low density water has highly selective sol- solutions and gels, however, water is sometimes
vent properties. Lightly charged, weakly hydrogen- prevented from moving to abolish a gradient in its
bonding polyamide gels, on the other hand, contain chemical potential between two regions of freely
principally low density water. Since both intra- exchangeable molecules: it must then adopt a
and extracellular matrices consist of mixtures of different strategy for approaching equilibrium.
polymers presenting to water both highly charged Figure 1 shows an example which has been dis-
regions of surface and weakly hydrogen bonding cussed in detail elsewhere (Wiggins, 1990, 1995b).
or hydrophobic regions of surface (proteins, poly- This represents a protein molecule in solution with
saccharides, mucins, proteoglycans, polynucleo- its counter-ions, both positive and negative. Both
tides) it is highly probable that they, too, contain the protein molecule and the counter-ions have
both abnormal populations of water molecules. kinetic energy but, since the mass of the protein is
This expectation has many consequences for so much greater than that of the ions, its movement
physiological and pathological states of cells, some through the solution is slower. The result is that
of which are outlined briefly in the following enough ions to neutralize the fixed charges on the
discussion. protein move through the solution with it. These
10656995/96/060429+07 $18.00/0 ? 1996 Academic Press Limited
430 Cell Biology International, Vol. 20, No. 6, 1996

+ +

+
+ 1 2

+ +
+


+
+
4 3
+
+

+ + +

Fig. 1. A protein in solution with its counter-ions. Fig. 2. Growth and shedding of high ( ) and low density water
( ) round a diffusing polyelectrolyte ( ).

ions can exchange rapidly with other ions in the


solution but, whether excess electrolyte is present
or not, there is always a higher local concentration
of ions in the restricted zone round the protein, the
double layer of fixed charges and counter ions, than 1 2
in the rest of the solution. It follows that there is
also a gradient of osmolality across the apparent
interface between that zone and the rest of the
solution. The high electric field at the surface of the
protein prevents water from moving to equalize 4 3
its activity in the two regions. Experiments with
gels (Etzler and Fagundus, 1983; Wiggins, 1995b)
have suggested that equilibration of water is
approached by means of changes in local density.
Fig. 3. Growth and shedding of low density water ( ) round a
Starting at that imaginary interface where hydrophobic molecule ( ).
molecules sense their state of disequilibrium and
moving away from it in both directions, the local
density of water increases in the double layer where
its activity is low and decreases in the rest of the of mp/mw, the ratio of the mass of the protein to the
water. Propagation of high density water is limited mass of a water molecule. Thus the larger the
by the protein surface; it seems probable that protein molecule the thicker the zones of modified
propagation of low density water is limited by water. Even in a gel the matrix has mobility, even if
relative rates of diffusion and by convection and only of vibration of side chains, so that the extent
stirring. of development of high and low density water is
If the protein molecule in contact with water limited. The limitation of growth of modified water
were absolutely stationary, high and low density layers is illustrated in Fig. 2.
water zones would grow out from its surface with A similar argument applies to hydrophobic areas
progressive movement of water molecules together of protein surfaces and hydrophobic moieties of
in the double layer and apart in the external water, other molecules, at which water molecules can
generating, at the rate of self-diffusion of water, make fewer and weaker hydrogen bonds than mol-
larger and larger zones of high and low density ecules in the bulk. Collectively these molecules
water. Their size would then be limited by convec- move apart from one another, decreasing their
tion currents, stirring etc. The protein, however, density to compensate for their increased enthalpy,
is not stationary, and as it moves through the generating a zone of low density water (see Fig. 3).
solution relatively slowly it sheds its waters of In this case the maximal limit to growth of low
hydration, which immediately revert to normal density water may be set by the distance over which
density while other waters are freshly perturbed by hydrogen bonding is cooperative (Barnes et al.,
the protein surface. These dynamics put an upper 1979). This maximum may not be reached for a
limit on the average thickness of modified water small molecule with a high diffusion coefficient,
zones associated with the charged regions of the but may be approached by a larger less mobile
protein surface. Presumably, it would be a function hydrophobic surface.
Cell Biology International, Vol. 20, No. 6, 1996 431

+
+ +
+
+ + +
+
+
+ + +
+ + + + + +

+ + + + +
++ +
+
+ +

+
+ +
+
+ + + +

Fig. 4. Aggregation of two hydrophobic molecules in solution: Fig. 5. Aggregation of two polyelectrolyte molecules ( ) in
the hydrophobic interaction. ( =hydrophobic molecule; solution: the micro-osmotic force. ( =high density water;
=low density water.) =low density water.)

Effects of solutes A similar force for aggregation has been ident-


ified in charged biopolymers (collagen triple helices
The highly selective solvent properties of high and and DNA double helices (Rau and Parsegian, 1992;
low density water also affect their lifetimes. When Leikin et al., 1994) osmotically stressed by poly-
solutes partition selectively into low density water ethylene glycol. It has been suggested (Wiggins,
(K + , univalent anions, amino acids, compatible 1995b) that this force, which also increases with
solutes) it increases in volume, if water can move to increasing temperature, is due, not to a direct
follow the concentrating solute, or it reverts toward interactive force between polyelectrolytes, but due
normal density; solutes relatively excluded from to movement of water outward from between poly-
low density water (small cations, hydrophobic mol- electrolytes. Parsegian and coworkers have called it
ecules) decrease its volume but decrease its density the hydration force; it appears to be the same as the
still further (Wiggins, 1995b). At a charged inter- micro-osmotic force described here. Figure 5 shows
face, if low density water accumulates enough two polyelectrolyte molecules, each surrounded by
solute to allow it to relax back to normal density, a region of water of low activity, containing the
high density water also relaxes. counter-ions in contact with a region of relatively
high water activity; the system can lower its free
Hydrophobic and micro-osmotic forces for energy either by expansion of water in the zones
assembly of biopolymers where its activity is high or, as in the hydrophobic
interaction, by eliminating some high activity
The hydrophobic interaction has been interpreted water. In both Figs 4 and 5 the aggregation is such
in terms of the growth of low density water at that the surface area of polymer in contact with
hydrophobic surfaces (Wiggins, 1990); it is illus- water and therefore the volume of residual high
trated in Fig. 4. Each of two separate hydrophobic activity or high enthalpy water is minimized. The
molecules has a zone of high enthalpy water sur- three forces which determine whether polyelectro-
rounding it. The solution can decrease its overall lyte molecules aggregate or not (electrostatic
free energy in two ways: either two hydrophobic repulsion, van der Waals attraction and the
surfaces align themselves to each other, driving out micro-osmotic force) are additive.
some of the high enthalpy water, or the high Figure 6 illustrates some extremely simple exper-
enthalpy water expands, decreasing its local density iments with solutions of sodium dextran sulphate
and chemical potential. Each of these solutions has (molecular weight 500,000) which undergo phase
its problems: expansion of water is opposed by the separation as a result of the operation of the
structural entropy term TS; expulsion of water micro-osmotic force. A solution of dextran sul-
from between hydrophobic surfaces increases the phate of about the same water content as a cell or
entropy of water but decreases that of the solute extracellular matrix (3 g water/g dry weight) is
molecules. extremely viscous; addition of NaCl, of any con-
Figure 4 shows a compromise state of lowest centration, decreases it viscosity but maintains it as
overall free energy in which some water has been a single solution. Addition of KCl above a critical
expelled, decreasing the amount of high enthalpy concentration (0.7 at 21)C) induces separation
water and the residual water has expanded. into a clear viscous gel containing most or all of the
432 Cell Biology International, Vol. 20, No. 6, 1996

M KCl
NaCl

M NaCl

Two phases Dextran sulphate Single solution


Top: fluid in water fluid
Lower: gel single solution
viscous

KCl KCl

NaCl NaCl
NaCl NaCl

Fig. 6. The micro-osmotic attractive force, driven by KCl and the repulsive micro-osmotic force, driven by NaCl in dextran
sulphate solutions. ( =dextran sulphate; =high density water; =low density water.)

dextran sulphate and a fluid watery phase; i.e. This is an example of relaxation of high density
dextran sulphate molecules have aggregated into water back to normal density as it is no longer
the gel phase and expelled water. This is the stressed by contact with low density water. These
attractive micro-osmotic force. It is opposed and simple experiments show that because the micro-
the single phase restored by addition of a suitable osmotic force is not a direct force between one
concentration of NaCl; this is an example of the macromolecule and another it can be either attrac-
repulsive micro-osmotic force. tive or repulsive, depending upon the direction of
The two lower boxes illustrate the roles of the the osmolality gradient down which water moves.
two populations of water molecules. When NaCl is The direction of that gradient, in turn, depends
added to a solution of dextran sulphate in water it critically upon the small solutes present. It is pro-
partitions selectively into high density, low activity posed that this versatility, combined with a cells
water, generating a fresh osmolality gradient which unique ability to control its cytoplasmic compo-
is partially abolished by movement of water from sition by means of pumps and channels is an
its low density region into the high density region, important component of metabolism and homeo-
thus converting some low density water into high stasis. It must also be an important factor contrib-
density water. Addition of KCl to the solution of uting to cellular volume regulation (Macknight
sodium dextran sulphate in water, on the other et al., 1994).
hand, converts some high density water into low
density, high activity water. Two possible responses
of the system in its approach to its state of lowest THE STRUCTURE OF THE CYTOPLASM
overall free energy are: on the left, it squeezes out
some of the high activity water, yielding two There is considerable evidence that cytoplasm is
phases, the extruded water on top and a gel, still not a simple solution with proteins, electrolytes and
containing some low density water, at the bottom; non-electrolytes all randomly dissolved in all the
on the right all high activity water has been water (Pagliaro and Taylor, 1988; Getzenberg
extruded and the gel contains only normal water. et al., 1991; Knull, 1992; Luby-Phelps et al., 1993).
Cell Biology International, Vol. 20, No. 6, 1996 433

+ + + + + + + + + + + +
+ +
+ + + +
+ + + + + + +
+ + + + + +
+ + + +
+ +
+ + + + + +
+ + +
+ +

+ + + +
+ + + + + + + + + + + +
+ + + + + + +
+ + ++
+ + + + +
+ + + + + +
+ + + + + + +
+ + + + + + + +
+ + +
+ +
+ + + + +
+ + +
+ + +
+ + + ++ +
+ + + + + + + + +
+ + + + + +
+ +
+ + + + + + + +
+ + + + + +
+
+ + + + + + + +
+ + + + +
+ + + + + + +
+ + + + + + + + +
+ + + + + + +
+ + + + + + +

+
+ + + + + + +
+ + + +
+
+ + +
+ + + + + + +
+ + +
+ + +

Fig. 7. Proposed physiological structure of left, a quiescent cell and right, an active cell. ( =protein; =high density water;
=low density water; , =compatible solute.)

Al-Habori (1995) has reviewed evidence for the rest of cytoplasmic water which contains only low
microcompartmentation of the cytoplasm and concentrations of compatible solutes and few ions.
possibly metabolic channelling; i.e. the transfer of Responses to this state of disequilibrium are three-
the product of one enzyme reaction directly to the fold: first, some of the high activity water between
next enzyme in the metabolic sequence without proteins is squeezed out, reducing its volume and
an intermediate sojourn in solution in the main thus decreasing the overall energy of the cytoplas-
body of cytoplasmic water. Drost-Hansen and mic compartment; second, the water remaining
coworkers have written extensively about the between double layers decreases its local free
possible role of vicinal water in cellular processes energy by expanding; third, water in the double
(Clegg and Drost-Hansen, 1991; Drost-Hansen layers increases its local free energy by compacting.
and Singleton, 1992a,b). Clegg (1984, 1992) has In its final state the cytoplasm consists of proteins,
suggested that enzymes are clustered round the surrounded by high density, fluid, reactive water
actin filaments of Porters (1984) microtrabecular containing most of the ions of the cytoplasm,
lattice; he has shown that removal of much of the clustering round actin filaments in such a way that
rest of the water from the cytoplasm has little effect their surface areas are minimized, so that the
upon enzyme activity. Cleggs perception of the amount of residual high activity water which
structure of the cytoplasm is consistent with much expands and becomes viscous and inert is mini-
of the experimental data both from his laboratory mized. This is the quiescent state of a cell illustrated
and from others. Figure 7 illustrates a modification at the left of Fig. 7. The active state of a cell is
of his concept, incorporating the micro-osmotic represented at the right of Fig. 7. Cells are acti-
principles outlined above. vated by influx of ions (Ca2+ and Na + ) which
Intracellular proteins are highly charged and, as induce the repulsive micro-osmotic force, increas-
illustrated in Fig. 1, are surrounded by a zone of ing spaces between proteins, increasing the amount
water in which the counter ions (mainly K + and and fluidity of high density water. This usually
Cl " are confined. In the resting state of a cell Ca2+ stimulates cascades of phosphorylation of proteins
is sequestered in intracellular stores and Mg2+ (Torphy, 1994), which, by increasing charges on
complexed to ATP and other anions. The immedi- their surfaces increase still more the amount and
ate effect of the high local concentration of ions fluidity of high density water and decreases the
surrounding the proteins is to create a water amount, but not the viscosity of low density water.
activity gradient between the double layers and the Although transfer of a phosphate from ATP to a
434 Cell Biology International, Vol. 20, No. 6, 1996

+ + + + + + + + + + +

+
+ +
+ +

+
+ ++

+
+

+
+ +

+
+ + + + + + + ++ + ++
+ + +

+
+

+
+ + + + + +
+ +

+
+ + + + +
+ + +
+ + +
+ +

+
+

+
+ + + + +

+
+ + + + + + + + + +
+

+
+ + + + ++ + +

+
+
+ + +

+
+ +
+ + + + + + + +

+
+

+
+ + + ++ + +
+ + + + +
+ + + +

+
+ + + + +

+
+ + + + +

+
+
+ + + + + + +
+ + +

+
+ + + + + +
+

+ +

+
+ + + ++
+ ++

+
+ + +
+ + + + + + +
+ +

+
+ + +

+
+ + + + +

+
+ + + + +
+

+
+
+
+ + + +

+
++ + + + +

+
+
+ + + + +
+

+
+
+ + + + + + + +
+ + + +

+
+ +

+
+ + + +
+ + +
+ + + +

+
+

+
+ + + + + + + +

+
+
+ + + + + + + + + + +

+
+ + + + + +
+ + + + + +
+ + + + +

++ + + +
+ + +
+ + + + + +

+ +
+ + +

+
+ + + +

+
+ + + + +
+

+
+

+
Fig. 8. Two proposed pathological states of a cell: left, a rigid state of rigor; right, a hyperactive uncontrolled state. ( =protein;
=high density water; =low density water; =compatible solute; # =carcinogen.)

protein does not increase the number of negative Polymers are plasticized by water and with too
charges in the cytoplasm it does increase the little water present they assume a state in which any
number of fixed charges on polymeric surfaces; as movement is extremely slow (Slade et al., 1993).
pointed out earlier, the size of the molecule carry- Residual water associated with the proteins is of
ing a charge is important in determining the extent two extreme forms. The high density water is very
of growth of high and low density water popu- fluid and very reactive because it is rich in free OH
lations. The average viscosity in the cytoplasm of groups and lone pairs of electrons, which are
the active cells is so lowered that diffusion is rapid, the reactive centres of water molecules. It has been
resistance to conformational changes of proteins suggested that this might be a contributory mech-
reduced, and gross protein movements (cytoplas- anism of apoptosis in which sustained increases in
mic streaming, sliding of filaments, etc.) facilitated. intracellular Ca2+ levels lead to fragmentation of
Moreover, there is a fast track through extremely DNA, plasma membrane disruption and irrevers-
fluid water which will selectively retain most ions ible depletion of ATP (Santos-Argumedo et al.,
and many metabolites. 1993). The extreme reactivity of high density water
The active state relaxes back to the quiescent has been demonstrated in unpublished experiments
state when the stimulating ions are pumped back with Dowex AG 1X8 anion exchange resin. The
across the plasma membrane or into cytoplasmic charged groups -N(CH3)3 + are hydrolysed in
stores. This reversal of the repulsive micro-osmotic water, the rate of production increasing with
force restores the tightly clustered quiescent state. conditions which promote high density water.
Figure 8 illustrates two possible pathological An entirely different possible pathological state
states of cytoplasm. Experiments with ion exchange is illustrated on the right of Fig. 8. Carcinogens
resins have shown that Ca2+ ions are the preferred are often either small hydrophobic molecules or
counter-ions at negatively charged surfaces cations, such as Ni2+ . Both species accumulate
(Ca2+ >Mg2+ >K + >Na + ). They induce extreme selectively into high density water and their
forms of high and low density water. The phenom- removal can pose problems. Cells have highly
enon of rigor in muscle occurs when ATP is selective and efficient transport mechanisms for
exhausted and Ca2+ cannot be transported back removal of ions which they commonly encounter
into the sarcoplasmic reticulum. It is a state of and use like Na + , H + and Ca2+ ; they are not likely
extreme and irreversible rigidity (Grinwald and to be as effective for foreign ions like Ni2+ . Small
Nayler, 1981). The left of Fig. 8 illustrates this. The hydrophobic molecules have been shown to have a
extremely high relative activity of water between remarkably high affinity for high density water
double layers results in expulsion of much water from which they are almost impossible to wash out.
with formation of a low water content rigid gel. For example (Wiggins, 1995b), BzOH disappeared
The rigidity of the gel is due more to its low water entirely into both a cation and an anion exchange
content than to the absolute viscosity of the water. resin and could not be eluted with 10 bed
Cell Biology International, Vol. 20, No. 6, 1996 435

volumes of water. It was finally removed following D-H W, S JL, 1992a. Our aqueous herit-
neutralization of the gel. A plasma membrane is no age: evidence for vicinal water in cells. In Fundamentals of
Medical Cell Biology, 3A. JAI Press, 157180.
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molecules and their high selective solubility in high heritage: role of vicinal water in cells. In Fundamentals of
density water is apparently not balanced by an Medical Cell Biology, 3A. JAI Press, 181202.
efficient extrusion mechanism. Cells have only E FM, F DM, 1983. The density of water and
P-glycoprotein which is induced in drug-resistant some other solvents in narrow pores. J Colloid Interface Sci
115: 513519.
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K HR, 1992. Association of glycolytic enzymes with the
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state illustrated in Fig. 8 contains more water and of forces between self-assembled proteins: temperature-
more Na + ions than normal cells. Accumulation dependent exponential forces between collagen triple helices.
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ACKNOWLEDGEMENT 1993. Cytoarchitecture of size-excluding particles in living
cells J Cell Sci 106: 565578.
R DC, P VA, 1992. Direct measurement of
This work was funded by a grant from the Health temperature-dependent solvation forces between DNA
Research Council of New Zealand. double helices. Biophys J 61: 260271.
S-A L, T C, P G, K PA,
P RM, 1993. A B lymphocyte surface molecule
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