1

Life depends upon two kinds of water

by

Philippa Wiggins

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Chapter headings page

1. Introduction 4 2. Two-state water 6 3. The experimental strategy 13 4. Solutes and two-state water 18 5. Surfaces and two-state water 24 6. Summary of two-state water and surfaces 26 7. Solution and folding of proteins 35 8. Synthesis of ATP from ADP plus Pi in LDW

and its hydrolysis in HDW 37 9. Mechanism of sodium transport driven by ATP

and its hydrolysis 40 10. Ion channels 42 11. Preservative solutions 44 12. Formation of bone 49 13. Origin of chirality 52 14. Coupled reactions in biochemistry 60

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Chapter 1

Introduction As every biochemistry student knows, enzymes can do anything. With great specificity they catalyse all metabolic reactions; with the aid of small solutes such as ATP and cations, they reverse many spontaneous hydrolysis reactions and accelerate other reactions which, although spontaneous, do not take place in vivo in the absence of enzyme.

The ‘how’ of these manifold reactions is less obvious. Although the shorthand version of the hypothetical student of biochemistry invoked only enzymes, the operational entities are, in fact enzymes-in-water or, better still, enzymes-in-solution. The chemistry of the surfaces of proteins is well-known. They carry hydrophobic groups, weakly hydrogen-bonding groups such as -C-OH, >NH, -NH2 and charged groups which are the ionised forms of weak acids (-COO-) or weak bases (-NH3

+), and these charged groups are neutralised by counter-ions. The specificity of the attachment of substrates to such surfaces arises from geometrical matching of hydrogen bond donors and acceptors, of opposite dipoles and of opposite charges. It is hard, however, to imagine how the conformational changes that the proteins undergo during activity can transform these weakly-interacting groups into the aggressively reactive state required for some enzymic processes. Just four reactions have been chosen to illustrate this problem.

Energy transduction: the great biochemical myth

Michael King (2003)1 in his Penguin History of New Zealand described the Great New Zealand Myth. This was published in 1909 and thereafter in the School Journal, which was read by all primary school children in New Zealand. It described in detail how the great Polynesian navigator, Kupe, discovered New Zealand in 950 AD. Generations of New Zealanders have believed that this was authentically derived from Maori oral tradition. In fact it was pure invention by a non-Maori. A similar status is enjoyed by the biochemical concept of energy transduction which appeared in early text books2 and is believed by generations of biochemists to be in the great tradition of Willard Gibbs and other classical thermodynamicists, who must be in perpetual motion in their graves.

Energy transduction says that the free energy of hydrolysis of ATP or of dissipation of a cation gradient can be harnessed by enzymes to do work. In fact the free energy of any spontaneous reaction is always dissipated as heat. Reactions are coupled, in a thermodynamic sense, only when the product of one reaction is a reactant of the second. So the energy required to transport cations against gradients or to make the filaments slide or to synthesis ATP or peptides or polynucleotides or to perform any of the reactions idiosyncratic of life, must have another source. That hydrolysis of ATP is an essential part of the overall reactions of cation pumps and motor molecules is not in question, but the mechanism is. Since water is a reactant in these reactions, and is also the solvating medium in which they take place, its involvement is inevitable. Phosphorylation of the enzyme by ATP must change the local environment

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in which the reaction takes place, so that its free energy change becomes negative. That environment is water close to the enzyme surface.

Hydrolysis of peptides and polynucleotides.

Proteases and Dnases hydrolyse peptide bonds and oligonucleotide bonds, respectively, at neutral pH and 38oC or ambient temperature. To break these bonds in vitro requires boiling in 6M HCl. Somehow these enzymes-in-solution have acquired the properties of hot concentrated strong acids. Again it is impossible to ignore a contribution of water, which is present and is required for the development of acid properties.

Formation of bone

The mineral component of bone is Ca3(PO4)2. In vivo this forms at pH 7.4 and 38oC from the normal concentrations of calcium and phosphate (approximately 2 mM) in blood. Similar ceramics require a temperature of 1200oC, presumably, to dry the product. The concentration of the species PO4

3- in the blood is probably of the order of 10-6 M, making it far too dilute to precipitate out at all. Indeed, we know that the solubility product is not exceeded in blood because bone forms only in conjunction with specialised cells (the osteoblasts). Crystallisation of Ca3(PO4)2 is a process which depends entirely upon the solvent, which is water, and the electrostatic attraction between Ca2+ and PO4

3-. Only changes in the solvent properties of water could allow crystallisation of bone at pH 7.4 and 38oC. Sodium and potassium Many enzymes discriminate with exquisite precision between sodium and potassium, a feat which appears beyond the most green-fingered chemist. Carboxyl groups show a slight preference for K+ over Na+ (2:1) but nothing approaching the 30:1 of the Na,K-ATPase and the many other enzymes which absolutely require either K+ (eg pyruvate kinase) or Na+ (eg Na+-dependent active transporters). A scrutiny of the aqueous solution chemistry of the two cations shows that their only significant difference lies in their effects upon the water in which they are dissolved. Na+ slightly increases water structure and K+ slightly decreases water structure. Can water change its physical and chemical properties? Table 1 shows the boiling points of the hydrides of oxygen and its neighours in the 1st row and 6th group of the Periodic Table. Neighbouring hydrides in the first row are all gases at ambient temperatures; they boil well below 0oC, the freezing point of water. As one moves up the hydrides of Group 6a elements from Te to S, their boiling points progressively decrease. Extrapolating these boiling points to the molecular weight of water, gives an expected boiling point of about –75oC. These figures were first produced by Henderson (1913)3.

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Table 1: Boiling points of some hydrides in oC

Group 3a 4a 5a 6a 7a B2H6 : -92.5 CH4 : -164 NH3 : -33.4 H2O : +100 HF : -87.7 H2S : -60.7 H2Se : -41.5 H2Te : -2

There is obviously something very strange about the boiling point of water. We now know that the strangeness is a consequence of the extensive three-dimensional hydrogen-bonding of water molecules to eachother; breaking the residual hydrogen bonds in the liquid requires an abnormally high temperature. Reverting to the hydrides of the first row, however, we note that NH3 and HF, which also mutually hydrogen bond, still have much lower boiling points than does water. The explanation is that the strength of water-water hydrogen bonds greatly exceeds that of intermolecular bonds in NH3 or HF.

The question that heads this section, therefore, can be answered with a qualified yes. If water, somehow, locally changed its density so that its hydrogen bonds became straighter and stronger or bent and weaker, all its physical and chemical properties must change. Its boiling point would decrease as bonds became weaker, free energies of hydration of Na+ and K+ and of other cations and of reactants and products of hydrolysis reactions must all change. An increase in hydrogen-bond strength must lead to the opposite changes in physical and chemical properties.

Does water change its properties at surfaces ?

In order for these various processes to take place, the changes in the structure of water and, therefore, of its properties must extend over a zone large enough to accommodate the reaction species. Before the early 1990s no plausible mechanism for the formation of appreciable volumes of modified water had been suggested. Ling (1969)4 had recognised this problem and proposed that intracellular water existed as polarised multilayers at protein surfaces, thus providing adequate volumes of modified water. It has generally been considered, however, that hydration of a protein surface is not long-range. At a protein surface, some water molecules must interact directly to hydrate the surface, but there is no reason to expect that this would involve more that one or at the most two, layers of water molecules. The dominant water-water bonding must then take over, if water consisted, as was generally believed, as a single continuous three-dimensional hydrogen-bonded network in which water-water bonding was, in general, stronger than water-surface bonding5

In spite of this theoretical void, the clear need for mechanisms in the neglected areas of energy transduction, hydrolysis of peptides, mineralisation of bone and discrimination between Na+ and K+, encouraged a search for modified water inside cells and at surfaces. After all, experiment is the final arbiter. Both high density and low density water were found, but not consistently. In many experiments no change was detected in the properties of water at surfaces; when changes were detected they could only be interpreted in terms of an accepted model of water structure. That

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model was, usually, normal bulk-phase water together with water “bound” to surface moieties. This limited the usefulness of the experiments which had found modified water. The field was obviously ripe for an imaginative leap of understanding. But, of course, feasible leaps of understanding cannot be produced to order. The trick is to recognise one wherever and whenever it arises.

When it came, in the early 1990s, this imaginative leap came from physics, not biology6-12. It turned out to have the attributes of a good testable hunch. It was simple and limited in scope (proposing the coexistence of just two types of water in rapidly-exchanging microdomains). It offered many testable predictions, some of which satisfied the requirements for enzyme reactions already perceived; others were a bonus. For example, it was found that a solute which partitioned preferentially into low density water (LDW) (eg. K+) created local osmotic pressure gradients which could only be abolished by conversion of LDW to high density water (HDW). This immediately suggested a well-documented mechanism of enzymes. Enzymes start in an inactive state, are converted to an active state which performs the characteristic reaction, but must then revert to the inactive state in order to be able to repeat the cycle. If the active state contained LDW, then K+ would demolish it and restore the inactive state. This mechanism by which small solutes demolish their own preferred environment has proved of general application.

Two-state water and the problem reactions

With two-state water there is no need to invoke energy transduction. The activation of small cations and reversal of hydrolysis are inevitable consequences of the presence of LDW (see Chapters 8 and 14).

Behaviour as a concentrated strong acid is also an inevitable consequence of the presence of HDW (Chapter 8).

Crystallisation of bone becomes possible if Ca2+ and PO43- are first concentrated in

HDW and then precipitated when they convert HDW to LDW (Chapter 12).

Na+ partitions preferentially into HDW and induces LDW. K+ partitions preferentially into LDW and induces HDW (Chapter 4).

These manifestations of two-state water will be discussed in detail in what follows.

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Chapter 2

Two-state water Three independent lines of inquiry have converged on the proposal that

liquid water can exist as two different polymorphic structures. Such interdisciplinary convergence adds greatly to the plausibility of the proposal.

The model of GW Robinson and coworkers

Robinson and coworkers showed that all the anomalies of liquid water could

be accounted for quantitatively in terms of microdomains of different density6-8. Many of the anomalies, first listed by Henderson1 in 1913, have since been shown to be a consequence merely of the cohesive character of water; i.e. that water exists as networks of molecules interconnected by hydrogen bonds. For example the remarkably high melting point and boiling point of water can be explained in this way. When it is compared with other hydrides of Group 6 of the Periodic Table (H2S, H2Se, H2Te) which are not mutually hydrogen-bonded, the surprising prediction is that non-bonded water should melt at about –95oC and boil at about –75oC. In addition to these phenomena which are compatible with any cohesiveness, there is a group of anomalies which is more demanding. These have yielded to Robinson’s two-state water. They include a maximum in density at 4oC, a minimum in compressibility at 46.5oC and a decrease in viscosity with increasing pressure below 33oC. Moreover D2O and T2O differ from H2O more than predicted from cohesiveness alone.

Figure 1: A cluster of five water molecules as they would appear in ice1h or in LDW. Each oxygen is linked to two hydrogen atoms to which it is covalently bonded and to two more, on other water molecules, to which the central oxygen is hydrogen-bonded. In LDW each H atom lies on a straight line between two O atoms. HDW is a collapsed form in which the hydrogen bonds, which in LDW keep molecules apart, are bent and allow molecules to approach eachother and increase density. These bent bonds are relatively weak.

Figure 1 shows the structure of ice 1h and of LDW. In LDW each H atom lies

on a straight line between two O atoms. HDW is a collapsed form in which the hydrogen bonds, which in LDW keep molecules apart, are bent and allow molecules to approach eachother and increase density. These bent bonds are relatively weak.

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The model of HE Stanley and coworkers7-9 The most interesting region of liquid water lies in an experimentally

inaccessible temperature range below -40oC, the spontaneous nucleation temperature. Experimentally, it is impossible to prevent pure water from crystallising to ice. Computer simulations, however, can impose constraints that prevent freezing, so that the liquid survives to lower temperatures. At about -50oC, liquid water surpasses itself in strange behaviour and separates into two liquid phases of different density, a high density form and a low density form. Of course, this is computer water. It does not necessarily follow that real water would, if it could, do the same thing. But Stanley and coworkers have shown that as cooling continues, high and low density liquid waters pass smoothly into high and low density amorphous ices. These ices have been well investigated, both experimentally and in their pseudo electronic state. They really exist. Again, the estimates of density are about 0.91g/ml and 1.2 g/ml, a remarkable difference of 30%. The corollary of this separation into two liquid phases is that, with computer water, at least, warming through -50oC should reveal merging of the two liquids into one, so that, like Robinson’s model, microdomains would coexist to higher temperatures throughout the liquid range. Chaplin13 has suggested that water has an expanded and collapsed structures based on a 5-fold symmetry which limits growth of microdomains to about 3 nm diameter.

Evidence from biology

The third line of evidence comes from a scrutiny of biological processes, especially enzyme reactions. Like the outstanding anomalies of the pure liquid, a full mechanistic explanation seemed to require more than the random hydrogen bonding of one-state water. In particular, the ability of some enzymes to hydrolyse ATP, peptides and polynucleotides and others to synthesise ATP and biopolymers, a pervading phenomenon in biochemistry, had no detailed molecular mechanism. The concept of energy transduction which was proposed in the 1960s has survived to achieve the status of a textbook fact, but it does not constitute a molecular mechanism.

Before introducing this concept, the meaning of free energy changes, as used in biochemistry, needs clarifying. The free energy change of a reaction indicates how far the reaction, as written, is from equilibrium and, therefore, whether or not the reaction will proceed spontaneously. For example, the free energy change of the reaction

MgATP + H2O ADP + Mg2+ + Pi

is approximately –30 kJ/mol. Here Pi means the mixture of H2PO4-/HPO4

2- that exists at the prevailing pH. This is a rather high negative free energy change which means that the reaction, as written, will go spontaneously from left to right, and that the reverse reaction from right to left has a free energy change of +30 kJ/mol and will not go spontaneously.

The magnitude of the free energy change shows that the reaction is far from equilibrium. As the reaction proceeds from left to right, 30 kJ/mol of energy is dissipated as heat. Other biochemically relevant reactions with much higher negative

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free energy changes are hydration of most ions (eg K+, -352.0; Na+, -423.8; Li+, -529.4; H+, -1104 kJ/ml) (Tissandier et al, 1998)14. There are two important biochemical messages here. First, ions have great affinity for water and, secondly, when one talks about “bound ions” it must be remembered that dehydrating ions to free them to bind, say, to a carboxyl group, takes a very large amount of energy. Therefore, in the concentrations relevant to cellular solutions, ions tend to remain in solution, hydrated, and restricted in their movement because they must neutralise fixed charges on proteins. Such ions are not, strictly, bound but their mobility is linked to that of the protein.

The concept of energy transduction states that the negative free energy of a spontaneous reaction, such as hydrolysis of ATP, can be used to drive an uphill reaction, such as movement of cations from low to high concentrations, provided that the two reactions are coupled in a single enzyme active site (Biochemistry by CK Mathews & KE van Holde, 1990)15. The authors take the case where the uphill reaction has a positive standard free energy change:

A B ∆Go = +10 kJ/mol

The spontaneous reaction has a larger negative standard free energy change C D ∆Go = -30 kJ/mol

When the two reactions are coupled the overall reactions is: A + C B + D

and the free energy change is ∆Go = +10 - 30 = -20 kJ/mol

i.e. when the reactions are coupled, the sum of the two standard free energy

changes is negative, and the uphill reaction can proceed. The uphill reaction might be movement of Na+ from a low concentration inside a cell to a higher concentration outside the cell, and the spontaneous reaction hydrolysis of ATP. This is called energy transduction. It is taken to mean that free energy stored in the bonds of ATP can be converted to other forms of energy and harnessed to do work.

This concept was vigorously debated in the 1960s. Physical chemists pointed out that, however and wherever ATP was hydrolysed, the free energy of that hydrolysis was dissipated as heat and could not be diverted to or bestowed upon another unrelated reaction, however hungry that reaction might be for kJ16. True coupling of reactions requires that a reactant of the uphill reaction is a product of the spontaneous reaction, so that it is so rapidly scavenged that the reaction can proceed.

Biochemists, in their turn, pointed out that whenever the two reactions occurred together in the active site of the sodium pump, ATP was hydrolysed and work of transport performed. Without hydrolysis of ATP no transport occurred.

Resolution of the conflict

The solution to this controversy was clearly that both were partially right:

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that ATP was hydrolysed and that free energy dissipated as heat and that ATP hydrolysis was necessary for the work of transport to occur. But that the energy allowing the reaction to occur did not come from hydrolysis of ATP.

The first step in these transport reactions is phosphorylation of the enzyme by ATP17-19. It must follow that, by phosphorylating the enzyme, ATP induces a change in the local environment of the active site which allows the uphill reaction to proceed. This was a particularly good example to take, because, in the reverse running of the pump the same phosphoenzyme synthesised ATP from ADP, suggesting that the same changed environment allowed both the uphill Na+ transport and the uphill synthesis of ATP to occur. The debate, however, fizzled out, with the biochemists emerging as apparent winners by default.

A key paper by one of the groups participating in the debate, however, gave a very good clue as to the kind of local environmental change that phosphorylation of the enzyme caused. George et al (1970)16 showed that the free energy of hydrolysis of molecules like ATP was given by the difference between the free energies of hydration of products and reactants. They called this paper "Squiggle-H2O". An enquiry into the importance of solvation effects in phosphate ester and anhydride reactions. At this time the terminal phosphate bond of ATP was called a high-energy bond and given the symbol ~P. The "Squiggle-H2O" paper showed that there was nothing special about squiggle P but that the hydration free energies of all components of the reaction were of crucial importance. This immediately suggested a way out of the impasse reached between physical chemists and biochemists. Phosphorylation of the active site of the pump protein somehow changed the hydrogen-bonded structure of water inside that cavity. Changes in water-water bonding must change all free energies of hydration of solutes in that cavity. Some ions might move out of water which was less able to hydrate them (eg. Na+, H+, Ca2+) and both the sign and the magnitude of the free energy of hydrolysis of ATP might also change so that synthesis became a spontaneous process.

These changes in hydration free energies are necessary consequences of a change in local water-water bonding, because the first step in the solution of a solute is making a hole in the water big enough to accommodate it. This involves breaking a considerable number of water/water bonds. The free energy required to break bonds must change with the water structure.

At this stage the proposal that water in the enzyme active site assumed stronger water-water bonds when the enzyme was phosphorylated was highly speculative, and, of course, unpopular17-19. Unlike energy transduction, however, it was thermodynamically possible. Even at that time there were reasons to suppose that water at surfaces was perturbed relative to bulk water. Moreover, as more experimental data accumulated, the explanatory power of the hypothesis made it increasingly plausible.

A further indication that the energy for sodium transport did not come from ATP hydrolysis emerged later. Wiggins (1990)20 showed that transport occurred through the phosphorylated enzyme and was complete before the phosphoenzyme was hydrolysed; i.e. the uphill reaction was complete before the free energy of hydrolysis was released. It will be shown later that the hydrolysis of ATP is necessary to restore the enzyme to its initial resting state so that the cycle can be repeated.

ATP the energy currency of the cell

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ATP hydrolysis is associated with a very large number of reactions which cannot proceed spontaneously but do so when accompanied by ATP hydrolysis21-26. These include uphill transport of Na+, H+, and Ca2+, driving motor molecules, folding proteins, and synthesising peptides, polynucleotides and polysaccharides. It became clear that the change in microenvironment of these enzymes upon phosphorylation or binding of ATP must be of a very general nature. Ions transported uphill (Na+, H+, Ca2+) were all highly hydrated and of the class of ions which had long been known to increase water structure, whereas K+, which is pumped into rather than out of cells was a water-structure breaker. Other examples of “energy transduction” were synthesis of ATP (an uphill reaction) enabled by flux of cations downhill (a spontaneous reaction)26. In the ATP synthase the flux was of H+; in the reverse running of the Ca2+ pump the flux was of Ca2+ ions and in the reverse running of the Na+ pump, the flux was of Na+. Of course, the free energy changes of these spontaneous fluxes of cations were dissipated as heat. Again there must be a molecular mechanism whereby ATP is synthesised in the changed environment of the enzyme active site, and, perhaps released by a flux of cations.

The status of the concept of energy transduction in biochemistry is probably one consequence of the specialisation of scientists in the last half of the twentieth century. Langmuir, when asked, called himself a scientist, not a physicist or physical chemist. Pauling, though known as a chemist first and foremost made extensive contributions to biology. In the 1960’s, physical chemists, such as George16 , were familiar with the growing biological literature and could contribute with authority to the thermodynamics of active transport. Now, most physical chemists are not aware of the thermodynamic problems hidden in biochemistry; and biochemists learn their thermodynamics from biochemists.

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Chapter 3

The experimental strategy

The amount of water contained in the active sites of enzymes is so small that its direct characterisation was very difficult. Any water-related property must be swamped by the simultaneous presence of excess bulk water. Experiments with actomyosin, the Ca-ATPase of sarcoplasmic reticulum and mitochondria,however, are discussed elsewhere27.

.. A corollary of the proposal for water with stronger water-water bonds in enzyme active sites, however, was that such water must also exist elsewhere. Vital water, as a scientific explanation, was not thinkable. Our experiments, therefore, concentrated on non-living water-filled microporous systems with cavities 1-3 nm across lined with protein-like surfaces; i.e. with weakly hydrogen-bonding, hydrophobic and charged moieties.

Dense films of cellulose acetate

The films of cellulose acetate that we used carried two acetate groups for each three C-OH groups. The films, themselves, were 4-20 µm thick and their pores about 2 nm in diameter. The surfaces were predominantly uncharged so that ionic selectivity could not be attributed to ion-pair formation. The most important property of these films was that the internal water compartment was easily probed selectively (Wiggins and van Ryn, 1986)28. Strips of film were soaked in solutions of various chlorides or nitrates, removed, excess bulk water blotted off and the residual film weighed, dried at 110oC and reweighed to give water content. Solutes were then extracted with 0.1 M HNO3. We found that up to six days were needed for ions to reach a steady state between internal and external water. This, presumably, was because the cross-linked network of cellulose acetate was extremely slow to expand or contract and allow water and solutes to move in or out. Internal water was highly selective to ions. KCl and CsCl were relatively accumulated from low external concentrations but as the external concentration increased the internal concentration became equal to it. NaCl, LiCl, HCl, CaCl2 and MgCl2 were increasingly excluded from the internal solutions. Moreover the degree of their exclusion increased with external concentration. Pieces of film were also blotted and the OH-stretch band of the infrared spectrum of water measured.

Figure 2. The energies of the OH stretch band in water, in ice 1h, water inside

cellulose acetate membranes and in solutions of various salts inside cellulose acetate membranes

water ice water NaCl LiCl MgCl2 KCl CsCl3000

3100

3200

3300

3400

3500

membrane water bulk water iceSolution

Wav

e nu

mbe

r of

max

imum

(cm

-1)

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. These results were dramatic: internal water of films soaked in water, NaCl,

LiCl or MgCl2 solutions had the spectrum of ice 1h (maximum at approximately 3200 cm-1), whereas films soaked in solutions of KCl or CsCl had the spectrum of liquid water (maximum at 3400 cm-1).

We concluded that KCl and CsCl were selectively accumulated into the ice-like internal water and, at high concentrations made it revert to normal water (see Figure 7). NaCl, LiCl and MgCl2, on the other hand increasingly excluded from ice-like water as their external concentrations increased, were imposing an osmotic pressure gradient on internal water, causing it to decease in low density and increase its property of excluding ions.

That we were dealing with osmotic pressure gradients was confirmed by experiments which showed that internal KCl concentration could be increased by co-equilibration with an excluded solute, such as MgCl2, betaine or butanol. It appeared that destruction of the ice-like structure by KCl could be prevented if it was balanced by an excluded solute, making the activity of water the same in the two compartments ( Figure 3).

Figure 3: K+, partitioning selectively into LDW inside a cellulose acetate membrane does not convert LDW to HDW when it is balanced osmotically by external Mg2+ wich is excluded fom LDW.

Microporous polyamide beads

Polyamide beads were also a good experimental preparation (Wiggins and

van Ryn, 1990)29,30. Again, it was possible to probe a single compartment and obtain surface water properties in the absence of significant surface charges. The density of internal water in variously-sized pores was measured in density bottles and found to decrease as the pore diameter decreased from 6 nm to 1.8 nm. Density of internal water also decreased when beads were equilibrated with increasing concentrations of polyethylene glycol 20M which was too big to enter the pores. In a batch of the same size of pores, but with higher negative charge, density of internal water was found to increase with added MgCl2 (Wiggins et al, 1991).

Dextransulphate solutions Dextransulphate (MW 500,000) is a typical highly charged polyelectrolyte

with two sulphate charges for each three glucose monomers. It formed rather viscous

LDW K+ Mg2+

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solutions, except in the presence of KCl or CsCl when it separated into a gel, containing all the dextransulphate and a polymer-free electrolyte solution. We measured the effects of added solutes on the viscosity of solutions. NaCl, MgCl2 and butanol decreased the viscosity of dextransulphate solutions. Urea increased viscosity at low concentrations and decreased it at high concentrations. Betaine and trimethylamine oxide increased viscosity at all concentrations31.

Figure 4. Effects of solutes on the viscosity of dextransulphate solutions at concentrations of 1 g dextransulphate in 3 ml water. At higher concentrations of KCl dextransulphate precipirtated.

Hydrophobic glass beads

The dextransulphate experiments had given information about addition of

excess solutes to viscosity. Some of this effect might have been due to the change in the countercation and some to addition of excess ions. With the small glass beads31 we could change the countercation only so that we could separate that effect from the effect of added salt. Counter cations proved to have highly specific effects, especially when the rest of the glass surface was made hydrophobic by treatment with dimethyldichlorosilane: low density water was induced with K+, Cs+ and Ca2+ but not with Na+, Li+ or H+. Beads of all sizes (45 to 500 nm in diameter) adhered to each other and to the surfaces of tubes containing them, whether they were hydrophilic or hydrophobic. This stickiness was a feature only of beads in water and D2O but not in ethanol, methanol, DMSO or hexane.

The hydrophobic glass beads had the extraordinary property of floating on

water, despite their density of 2.6 gml-1. The floating ability and mutual adhesion of beads depended critically upon the counter-ion.

0.0 0.2 0.4 0.6 0.8 1.00.5

1.0

1.5 urea

KCl

NaCl

betaine

TMAO

[solute] (M)

rela

tive

visc

osity

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Figure 5. Glass beads (44-60 µm in diameter), made hydrophobic with dimethyldichloro silane. a, 2 g beads in 10 ml water; b, 4 g beads in 10 ml water with countercation K+. Cs+, Ca2+ or Mg2+. Floating of the beads of density 2.6 ml/g was due to trapping of gas molecules between the dry beads when surface LDW prevented their escape. Aggregation of the negatively charged beds was due to the need to minimise LDW at their surfaces. c, 2 g beads in 10 ml water with countercation Li+, Na+ or H+. Beads neither floated nor aggregated because their surfaces induced less LDW; d, the same as c photographed immediately after setting up; the haziness in the water shows the dropping of beads through the water.

Problems of interpretation

In one sense these experiments were in accord with the hypothesis that

water at protein-like surfaces could assume a low density strongly hydrogen-bonded state. But there were extreme difficulties in interpretation.

For example, K+ and Cs+, which are known to be chaotropes or water-structure breakers in simple solution, as countercations to glass beads, powerfully induced low density water. On the other hand, the known kosmotropes or structure-makers Na+, Li+, H+ did not. To add to the confusion, Ca2+ and Mg2+, two powerful kosmotropes, had effects similar to those of K+ and Cs+, both potent chaotropes..

Again, addition of excess chlorides of Na+, or Mg2+ decreased the viscosity of dextransulphate solutions, whereas, as kosmotropes they would be expected to increase it, by inducing low density water.

Urea, a favourite chaotrope of protein chemists, increased the viscosity of dextransulphate solutions at low concentrations and decreased it only at high concentrations. It also induced aggregation and floating of hydrophobic glass beads at low concentrations, and only at very high concentrations (6M) induced disaggregation.

Classical osmotic theory does not predict a change in density with the relatively low pressures encountered in osmotic systems, and yet imposition of an osmotic pressure gradient with polyethylene glycol 20 M outside polyamide beads decreased the density of internal water.

Both cellulose acetate, with predominantly hydrophobic, kosmotropic surfaces and microporous polyamide beads with predominantly hydrophilic, chaotropic surfaces, appeared to contain water enriched in low density water.

It was apparent that we were trying to interpret these experimental results in terms of an inadequately simple theory. The theory implicit in the thermodynamics of aqueous solutions assumes that all environments for solutes are statistically the same. This, however, changed with the proposal that water exists as a rapidly exchanging equilibrium between microdomains of different density. After 1994 and the

a b c d

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publication of Robinson’s modern version of a mixture model of liquid water (Vedamuthu et al, 1994)5 , it became possible to insert into this model the properties of water that had been uncovered in the experiments described above. In pure liquid water experiments are limited by the inability to probe a single type of microdomain, because their exchange is so rapid. This limitation, apparently, did not apply to the microporous systems we had used, in which it appeared possible to probe a single compartment. Such experiments had yielded characteristics of one type of enriched water, instead of the average properties obtainable in bulk water32-38.

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Chapter 4

Solutes and two-state water

Figure 6: LHS, single-state water in which all environments are the same; RHS, two-state water in which two very different environments coexist.

When a solute is added to single-state water, represented by the left hand

side of Figure 6, all environments are the same. But in two-state water there are two environments which are profoundly different, as model experiments have revealed. Any solute, therefore, partitions more-or-less differently between contiguous microdomains. The consequence is that there are instantaneous gradients of water activity between contiguous unlike microdomains. This is illustrated in Figure 7A.

.

Figure 7A. LHS, asymmetric partitioning of solute between LDW and HDW, creating local gradients in osmotic pressure; RHS gradients have been eliminated by conversion of LDW microdomains into HDW microdomains.

In Figure 7A the partition coefficient of the solute into LDW has been

arbitrarily given the value of 5. In a microdomain of 280 water molecules (Chaplin, 1999)13 this amounts to approximately a 1 molal solution. The difference between single-state water and this two-state water lies in the osmotic pressure gradients which the different solvent properties of the two types of microdomains inevitably create. This, presumably, was the reason for the impossibility of rationalising the

LDWHDW

LDW

5 1 5 5

5 5 5 5 5 5

5 5 5

1 1

11 1 1 1 1

11 1

3 3 3 3 3 3

3 333333 3 3 3 3 3

3 33333

HDW

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experimental results cited above. In two-state water there will always be potential local gradients in water activity. In solutions where there are no barriers to diffusion they must be avoided, eliminated or compensated for in some way.

Figure 7A shows how local gradients can be eliminated by converting LDW

microdomains into HDW microdomains until all concentrations are equal. In this configuration, however, water is in equilibrium but solute is not. The configuration arrived at by the solution of chaotrope cannot be illustrated by a drawing. It requires the methods of physical chemistry to elucidate the state of lowest overall free energy, taking into account the needs of water and of solute and of the system as a whole.

That configuration will include a reduction in the osmotic pressure gradients by conversion of some LDW into HDW with redistribution of solutes. That conversion, however, cannot proceed as far as that illustrated in the right hand side of Figure 7A both because it puts solutes out of equilibrium, but also because it involves a thermodynamic cost of displacing the water equilibrium so far. Moreover, if all water was converted to HDW by solution of a chaotrope, all chaotropes would have the same effect upon water structure. This does not happen. There appears to be a rank order of chaotropes and their effect upon the entropy of water.

A similar mechanism (Figure 7B) must apply to solutes which partition preferentially into HDW. In order to eliminate standing osmotic pressure gradients, some HDW microdomains must become LDW microdomains. Again, there will be a singular solution that will depend upon the magnitude of the partition coefficient, and on temperature and pressure. Figure 7B. LHS, asymmetric partitioning of solute between LDW and HDW, creating local gradients in osmotic pressure; RHS gradients have been eliminated by conversion of HDW microdomains into LDW microdomains.

Figures 7A and B are consistent with many observations that solutes which we had found partitioned into LDW (K+, Cs+, NH4

+, Cl-. Br-, I-, HCO3-, HSO4

-, H2PO4

-) are chaotropes in simple solution (Cacace et al, 1997) i.e. they decrease the structure of water or, in terms of two-state water, they induce HDW at the expense of LDW. Similarly solutes which partition preferentially into HDW ( H+, Li+, Na+, Ca2+, Mg2+, hydrophobic nonelectrolytes) induce LDW and are kosmotropes. The rank orders of positivity of the entropies of hydration of ions are the same as the rank orders of partitioning into LDW. (see Friedman & Krishnan (1973)39

HDW LDW

5 5 5

5 5 5

5 5 5

5 5 5

1 1 1

1 1 1

1 1 1

1 1 1

3 3 3

3 3 3

3 3 3

3 3 3

3 3 3

3 3 3

3 3 3

3 3 3

a b

20

This gives rise to a biological equivalent of Heisenberg’s Uncertainty Principle. Measurement of partitioning of solutes between HDW and LDW destroys the type of microdomain that they prefer. Therefore there are no neat lists of solutes and partition coefficients to delight the seeker after “hard numbers”. Partition coefficients depend upon all components of a system, including the concentration s of the solutes themselves.

Cost of displacement of the HDW/LDW equilibrium. An important secondary mechanism that emerges from the concept of

induction and elimination of osmotic pressure gradients in solution, is that displacement of the equilibrium in either direction incurs a thermodynamic cost. This follows because the position of equilibrium in pure water is set by the prevailing temperature and pressure. Its displacement introduces a positive term into the overall free energy of hydration. If this term is sufficiently large, the free energy of hydration may become positive and limit the solubility of the solute.

HDW LDW

From left to right: ∆S is negative, ∆H is positive, P∆V is positive From right to left: ∆S is positive, ∆H is negative, P∆V is negative

Since ∆G = ∆H - T∆S + P∆V, there are probably more instances of a

solubility being exceeded by a kosmotrope, which induces LDW, than by a chaotrope, which induces HDW. This is rather borne out by the extremely low solubility of hydrocarbons in water.

Partition of neutral salts

Figure 8: LHS Ca2+, a kosmotrope tends to partition into HDW, while Cl-, a chaotrope tends to partition into LDW. Separation of charge in a conducting medium is not allowed. RHS, the neutral salt has a slight preference for HDW.

The partitioning of single ions has been deduced qualitatively from the

behaviour of neutral salts in cellulose acetate membranes and polyamide beads28-38 Absolute values are not attainable, because the positive and negative ions of a salt in solution cannot be separated and their individual properties obtained. There is, however, another significant difference between single-state and two state water as solvents.

Cl- Ca2+- CaCl2

CaCl2

HDWLDW

21

This also follows from the differential solvent properties of the two types of water. Figure 8 shows that, since Cl- tends to partition into LDW and Ca2+ into HDW, each limits the concentration of the other, because electroneutrality within each microdomain must be conserved. There is a small relative accumulation of CaCl2 into HDW, requiring a low thermodynamic cost of conversion of HDW into LDW. CaCl2 has high solubility.

Figure 9: LHS, both Ca2+and SO4

2- are potent kosmotropes, both preferring HDW. RHS, this results in a much higher concentration of CaSO4 in HDW than in LDW, but also severely limits the solubility of CaSO4, because of the cost of converting so much HDW to LDW CaSO4 and CaHPO4, on the other hand, have extremely low solubilities. Although this is usually explained by a higher lattice energy of the ions in CaSO4 and CaHPO4 than in CaCl2, there must be a contribution from the high thermodynamic cost of hydrating salts containing both cationic and anionic kosmotropes. Since both ions partition preferentially into HDW the osmotic pressure gradient is considerable and there is a massive cost of induction of LDW. This is, perhaps, more obvious when the solubilities of ethane and ethanol in water are compared. Both ethane and ethanol have only weak intermolecular interactions. Nevertheless ethane is practically insoluble and ethanol extremely soluble. Ethane is a powerful kosmotrope with a high cost of induction of LDW. Ethanol consists of a combination of moieties of opposite characteristics. -C2H5, like ethane, tends to partition into HDW, but is opposed by -OH which tends strongly to partition into LDW. Since the whole molecule must partition one way or the other, the resulting osmotic pressure gradient is quite slight, and ethanol highly soluble.

Free energies of hydration of single ions

As pointed out above, free energies of hydration of single ions cannot be

measured directly because a single ion cannot be separated from its partner in a conducting solution except by the application of a high electric field. Nevertheless lists of free energies of hydration of single ions have been published. They are deduced from the experimental free energies of hydration of neutral salts, usually with some extrathermodynamic assumption. For example, it was often assumed that the hydration characteristics of K+ and Cl- were the same and that the free energy of hydration of KCl was equally divided between the two ions. With this assumption together with the further implicit assumption that the free energy of hydration of an ion was a constant quantity, independent of the identity of its companion ion or ions, free energies of hydration of other cations were calculated from the free energies of

CaSO4

HDWLDW

CaSO4

+ CaSO4 CaSO4

22

hydration of their chlorides and free energies of hydration other anions from their K+ salts. Tissandier et al (1998)14 in the values quoted in Chapter 2, avoided the first assumption but not the second. Two-state water seems to impose an extra constraint on single ion hydrations, which is illustrated in the comparision of the solubilities of CaCl2 and CaSO4.

What determines whether a particular solute prefers LDW or HDW ?

When a solute dissolves in water, there are three components of the free energy of hydration:

(i) water-water hydrogen bonds must be broken to make a hole into which the solute fits

(ii) water immediately adjacent to the solute molecule must reorient itself as it interacts with the solute molecule

(iii) the HDW/LDW equilibrium must shift so that water in contiguous microdomains is restored toward equilibrium.

Of these three terms, each increases in absolute magnitude as the volume of the solute increases and thus increases the extent of the interface between solution and water. ∆G1 is always positive as it involves breaking hydrogen bonds and increasing the entropy of water; it is greater for solution in LDW than for solution in HDW ∆G2 is the negative term which confers solubility upon the solute. It is of greatest magnitude for ionic kosmotropes; and of least magnitude for nonionic kosmotropes. ∆G3 is also positive because it involves displacing the water equilibrium at constant temperature and pressure and is of greatest magnitude for solution of kosmotropes in HDW. A remarkable feature, which is not intuitively likely, is that small ions with high surface charge densities and neutral hydrophobic molecules behave similarly. The odd solutes are large ions with low surface charge densities.

Biological solutes in two-state water Apart from ions, which can have extreme individual preferences for either

HDW or LDW, most biological solutes have relatively modest partition coefficients. This is because they have moieties which are chaotropic ( -OH, NH2, COOH), and moieties which are kosmotropic (CH in aliphatic and aromatic molecules). Since a molecule can be in only one place at a time, the overall preference for either HDW or LDW and the cost of abolishing the osmotic pressure gradient are both slight. These molecules are characteristically very soluble.

Salting in and salting out of proteins

Hofmeister first showed that some neutral salts increased the solubility of

proteins while others decreased solubility or were ineffective. The conventional view is that proteins and salts competed for water of hydration. The specificity of effects is more readily explained in terms of two-state water. A protein precipitates out when the thermodynamic cost of hydrating it becomes too high. Proteins are made up of

23

both kosmotroic and chaotropic moieties. If kosmotropic moieties predominate, precipitation of the protein will occur when the free energy of induction of LDW becomes too high. This could be offset by a chaotrope which induces HDW, decreasing the overall cost of displacing the water equilibrium. One would expect, therefore chaotropic solutes to increase the solubility of kosmotropic proteins and kosmotropic solutes to increase the solubility chaotropic proteins. This mechanism is compensatory rather than competitive. Like many phenomena of aqueous solution chemistry salting out of proteins is readily explained by the competition between solutes and proteins for water of hydration. Salting in, however, requires two-state water to explain how the solubility of a protein can be enhanced by addition of specific other small solutes

24

Chapter 5

Surfaces and two-state water Surfaces, also, induce osmotic pressure gradients in two-state water, since

chaotropic moieties on the surface take their primary hydration from LDW and kosmotropic moieties take their primary hydration from HDW. So far, surfaces are similar to solutes. In addition, however, surfaces induce osmotic pressure gradients in several modes that are not operative with small solutes. This leads to new levels of complexity.

Biological surfaces and two-state water

Biological surfaces are never smooth on an atomic scale. For example,

proteins have side-chains extending from the backbone of the molecule into surrounding water. A zone of water immediately adjacent to the backbone and containing those sidechains, has, consequently, a lower activity than a zone of similar size in the bulk solution. Water cannot move in toward the surface to abolish this water activity gradient, because the side-chains are covalently bonded to the backbone. This is an example of a compensatory mechanism in which the gradient of activity of water cannot be abolished by movement of water. As in classical osmotic theory, if there is a residual osmotic pressure gradient there must be a pressure gradient. In this case the pressure gradient is caused by water itself which tends to move in to abolish its activity gradient and is prevented. The pressure gradient, therefore is always directed inward toward the surface, so that there is a positive pressure acting on the water at the surface and a negative pressure acting on more distant water.

Figure 10: The roughness of a protein surface. Water immediately at the surface, between sidechains has lower activity than more distant water, The resulting osmotic pressure gradient becomes a pressure gradient with positive pressure at the protein surface and negative pressure further out, These pressures displace the local water equilibrium

The important difference from classical osmotic theory is that in two-state

water pressure gradients displace the water equilibrium, either inducing HDW where the pressure is positive and/or inducing LDW where the pressure is negative. This is illustrated in Figure 10. Whether the compensation for the osmotic pressure gradient predominantly induces HDW or LDW depends upon the position of the equilibrium before application of the gradient. If water at the surface is already predominantly HDW, then the pressure gradient will induce principally LDW. This is a source of

+p-p

+p

+p

+p

25

many apparent paradoxes. At high temperatures or high pressures when the bulk of water is principally in the form of HDW, surfaces tend to induce more LDW.

Charged surfaces and two-state water

A charged surface has an excess concentration of counterions in a zone

adjacent to the surface, creating another osmotic pressure gradient, which, again, cannot be abolished by movement of water, but acts as a pressure gradient oriented in the same direction as the gradient due to surface roughness. Again, it is independent of the chemistry of the surface moieties, other than their charge, and of the chemistry of the counter-ion.

The fixed charges on biopolymers are always strong chaotropes (large univalent anionic or cationic groups), inducing HDW in the double layer.

Experiments with glass beads (which will be discussed below) showed that counterions to fixed charges had powerful and extremely specific effects upon local water structure.

Added solutes also have effects other than that of the counter ion. Excess solutes can change the thickness of the double layer by changing the dielectric constant. When the double layer increases in thickness the excess concentration of counterions decreases, as do all the secondary effects of that osmotic pressure gradient. Conversely a decrease in its thickness amplifies all the osmotic effects and their consequences.

Superposition of the several compensations for osmotic pressure

gradients The five different modes of compensating for standing osmotic pressure

gradients sometimes reinforce one another and sometimes oppose one another. The two gradients which are essentially properties of the surface and independent of the chemistry of that surface always tend to induce HDW immediately adjacent to the surface and LDW further out. The gradient due to the specific chaotropic properties of the fixed charges has the same effect. When the counterion is also a chaotrope the cumulative effect is such that water adjacent to the surface appears to become pure HDW and compensation for the pressure gradient consists predominantly in induction of LDW. This, also, leads to many paradoxical observations. For example chaotropes can act as kosmotropes and vice versa. Some of these complexities are outlined in the next chapter.

26

Chapter 6

Summary of two-state water, solutes and surfaces

i) Ions that partition into LDW (K+, Rb+, Cs+, NH4+, C(NH3)4

+, Cl-, HCO3-,

H2PO4-, HSO4

-). In bulk water As illustrated in Figure 7, these ions all induce HDW: they are chaotropes.

They are large, univalent cations and large, univalent anions. The size of the cation appears to be dominant, because N(CH3)4

+, which has a considerable hydrophobic component, is a powerful chaotrope. The larger the anion or cation, the greater the asymmetry of partitioning.

At uncharged surfaces

Figure 11: a, a chaotrope partitions into LDW; b, water follows it to eliminate the osmotic pressure gradient; c, the cavity has reached the limit of its expandibility, and the osmotic pressure gradient is eliminated by conversion of LDW to HDW with exit of the chaotrope.

In pores of dense cellulose acetate films and small pores of polyamide

beads, pairs of such ions, of which KCl is the prototype tended to accumulate into LDW. If the cavity containing LDW was flexible, their accumulation was accompanied by water and the cavity swelled. This must often be the conformational change that is observed during enzymes function. .In a rigid cavity or in a flexible cavity which has reached the limit of its capacity to swell, further accumulation of KCl creates an osmotic pressure gradient which is eliminated by induction of HDW inside the cavity. As in Figure 7, KCl has now destroyed its own preferred environment and diffuses out, eliminating the osmotic pressure gradient. LDW reforms inside the cavity and the cycle is repeated. The frequency of these oscillations depends upon the flexibility of the walls of the cavity and also on the presence or absence of solutes which, by virtue of size or solvent selectivity are excluded from the cavity. In cellulose acetate membranes28 this was observed in two ways: The OH-stretch band of internal water was ice-like ( 3200cm-1) in the absence of KCl and water-like (3400 cm-1) in its presence at only 10 mM.. The concentration of KCl in

a b c

LDW HDW

27

the films at equilibrium was practically the same as that in the external solution. Presumably, the internal water was LDW before KCl was added and reverted transiently to HDW with KCl. It is significant that the OH-stretch band in the presence of KCl was that of normal bulk water and not either HDW nor LDW; i.e. it spent part of the time as LDW (a, Figure 11) and part of its time as HDW (b, Figure 11). The time average was the same as that of bulk water in which the OH-stretch band of water is the spatial average of LDW and HDW.

The local destruction of LDW by chaotropes at surfaces makes their experimental detection and characterisation extremely difficult. Strategies developed to over come these problems will be discussed later.

Such oscillations were observed in microporous polyamide beads when the solution bathing them contained both chaotropes (to cause swelling) and kosmotropes to reverse swelling32 .

Figure 12: oscillations in the internal volume of microporous polyamide beads

immersed in a Ringer solution containing chaotropes (K+, Cl-, H2PO4- and HCO3

-) causing swelling and kosmotropes (Na+, Ca2+ and Mg2+) becoming concentrated outside the pores and reversing the swelling. The periodicity of these oscillations was of the order of days because the cross-linked porous structure was rigid and slow to adapt to changing volume. Oscillations occurred only in the presence of both chaotropes and kosmotropes; beads swelled as chaotropes moved selectively into LDW in the pores. As they did so the kosmotropes in the external solutions, excluded from LDW, became more concentrated until the osmotic pressure gradient created by uptake of chaotropes reversed and beads shrank. In spite of appearances to the contrary, this is not perpetual motion because experiments were carried out at constant temperature, so that there was continual exchange of energy with the environment.

As counterion to a charged surface

28

As counterions these chaotropic ions became, paradoxically, powerful inducers of LDW. As they are lightly hydrated, their residual charge is high and they are held close to the fixed charge on the surface. This thins the double layer, increasing the osmotic pressure gradient due to the excess concentration of counterions.

Figure 13: a, Chaotropic fixed charges on the surface induce HDW which is already enhanced by HDW induced by surface roughness; b, counterions decrease the activity of surface water which, therefore, is further enhanced in HDW; c, the chaotropic properties of K+ as countercation cannot promote HDW further, because the equilibrium is already displaced to maximise HDW. The effect of this added chaotropic force is to induce LDW very strongly.

Their chaotropic property of inducing HDW in the double layer adds to

the chaotropic effects of the fixed charges, the chaotropic effects of excess counterion concentration and the chaotropic effect of surface roughness. These cumulative effects saturate the water in the double layer with HDW and force the residual pressure gradient to be satisfied by induction of a large zone of LDW outside the double layer. Hydrophobic glass beads31 with K+ or Cs+ as countercation floated on water and on D2O but not on ethanol, hexane, acetone or methanol. This presumably arose because the zone of LDW prevented escape of the kosmotropic gas molecules trapped between the dry beads. When beads were hydrophilic they did not float on water because air escaped into the neighbouring HDW.

The thermodynamic cost of inducing such an excess of LDW acted as a driving force for aggregation of such charged surfaces. This, too, was illustrated in the behaviour of hydrophobic glass beads31 which, with K+ or Cs+ as counterion aggregated strongly. These were remarkably powerful effects.

Dextran sulphate ( MW 500,000) in its Na+ form is highly soluble in water. Addition, however, of KCl or CsCl caused immediate phase separation of the solution into a gel of low water content containing all the dextran sulphate and most K+ or Cs+, and a simple electrolyte solution31 Again, this was, presumably, a way of avoiding the high cost of displacing the water equilibrium toward LDW.

-

-

- -

-

-

-

-

-

- -

- +p -p

a b c

K+

K+

K+

K+

K+

K+

K+

K+

29

As excess electrolyte

Excess KCl or CsCl had additional effects as the cation had its effect as a

counterion and the pair of ions was selectively accumulated into LDW outside the double layer, partially offsetting the cumulative osmotic effects which induced LDW. These neutral salts, therefore had dramatically non-linear effects upon water structure in dextran sulphate solutions (see Figure 4).

Univalent cationic kosmotropes (Na+, Li+, H+ ) In bulk water These small cations, with high surface charge density, partition into HDW

and induce LDW. H+ , with the highest surface charge density is the most powerful and Na+ the least.

At uncharged surfaces They are excluded from the predominantly LDW in dense films of cellulose

acetate and in small-pored polyamide beads, their partition coefficients decreasing with increasing with concentration. i.e. their exclusion creates an osmotic pressure gradient between water in the pore and external water and induces more LDW inside the pore. This can be used to increase the retention of a chaotropic solute, by balancing the excess osmotic pressure due to accumulation of chaotrope with an osmotic pressure due to exclusion of kosmotrope (see Figure 3).

As countercation at charged surfaces

Figure 14: the same charged surface as in Figure 13, but with Na+ as countercation. a, HDW induced by surface roughness and chaotropic fixed charges; b, Na+ as countercation is held further away than was K+ because it is more strongly hydrated. The double layer is, therefore thicker than in the presence of K+, and induction of HDW to compensate for the excess concentration of solutes is less; c, Na+ as a kosmotrope induces LDW in the double layer. The cumulative effect of all these factors is that Na+ induces very little LDW and appears as a chaotrope.

-

-

- -

-

- -

-

-

- -

- +p -p

a b c

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

30

As countercations to negatively charged surfaces, these highly hydrated cations have lower residual charges than the lightly hydrated chaotropic cations. They are, therefore, held further away from the fixed charges, increasing the thickness of the double layer and decreasing the osmotic pressure gradient due to excess concentration of cations. Moreover, because of their kosmotropic properties, they oppose the cumulative effects of surface roughness, excess counterion concentration and chaotropic fixed charges, so that water immediately adjacent to the surface is less enriched in HDW and much less LDW is induced outside the double layer. All these effects increase going from Na+ to H+. With Na+ , Li+, or H+ as countercation, charged surfaces show no tendency to aggregate. This is, perhaps, the most important difference between Na+ and K+ in cellular chemistry.

As excess electrolyte Excess NaCl partitions into HDW in the double layer, shielding counterions

from fixed charges, increasing the volume of the double layer and decreasing the osmotic pressure gradient created by excess counter ions. NaCl also increases the concentration of solutes in the double layer, causing further influx of water and increasing HDW at the expense of LDW. Thus, NaCl decreased the viscosity of dextransulphate solutions.

Figure 15: effect of excess NaCl, butanol and betaine on the relative viscosity of 10% dextransulphate.

These were large effects: 1 M NaCl halved the viscosity of the solution.

This figure also illustrates the point that highly hydrated salts (NaCl) and hydrophobic solutes (butanol) behave similarly in their effects upon water in the double layer. A metabolic consequence of this behaviour of Na+ is that its influx increases the fluidity of the intracellular environment and promotes cellular activity38.

Hydrophobic glass beads, with Na+, Li+ or H+ as countercation, neither

floated nor aggregated. (see Figure 5). This is particularly remarkable with H+ when

10 % dextran sulphate

0.0 0.2 0.4 0.6 0.8 1.00.2

0.6

1.0

1.4

BuOH

NaCl

betaine

solute (M)

rela

tive

visc

osity

31

the ionisation of the fixed charges must have been reduced, decreasing the electrostatic repulsion between beads.

Divalent anions and cations (Ca2+, Mg2+, SO4

2- and HPO42-)

In bulk solution These ions partition strongly into HDW and strongly induce LDW: they are

powerful kosmotropes. At uncharged surfaces They behave similarly to kosmotropic univalent cations; they are strongly

excluded from LDW in pores and cavities and can be used to balance the excess osmotic pressure of chaotropic solutes which partition into LDW.

At charged surfaces The remarkable power and specificity of divalent cations in enzyme

reactions will be discussed in more detail later. They most probably arise from the behaviour of the ions as countercations. Ca2+, for example is required for many protease reactions and for muscle contraction; Mg2+ is absolutely required for delivering ATP as MgATP to enzyme active sites. It is unlikely that these cations are often bound to carboxyl groups or phosphoryl groups on bioplymers because their free energies of hydration are so extremely high. Moreover they are present at very low intracellular concentrations, indeed. Furthermore it is unlikely that a single divalent cation is often able to neutralise two fixed negative charges which are usually too far apart. Ca2+ and Mg2+ form chelates with EDTA, EGTA, citrate and ATP, for example. Here two negative charges are close enough for a single divalent cation to neutralise them both, and the cations move through solution with their chelating agents. As counterions to fixed charges, on the other hand, each cation can usually neutralise only a single fixed charge. Since electroneutrality must be maintained in the smallest conducting compartment, each Mg2+ and Ca2+ in a double layer must be accompanied by a univalent anion. This doubles the osmotic pressure gradient across the interface of the double layer, because, the number of ions in excess of those in the rest of the solution is doubled. Therefore, without binding, one expects that divalent ions should have profound effects upon water inside and outside the double layer.

Divalent cations (Mg2+ more than Ca2+) have an extremely high affinity for

HDW. This, together with their double positive charges, makes them preferred countercations to the chaotropic fixed charges; they are held closely to the charges, so that the double layer is thin and the osmotic pressure gradient due to excess cation plus anion extremely high. Moreover, while the cations are both kosmotropes, their local effect upon the water equilibrium is moderated by the chaotropic anion that they carry in to maintain electroneutrality. The resulting configuration of the water inside and outside the double layer is illustrated in Figure 16, where it can be compared with those in the presence of Na+ and K+ as countercation. The combination of an extremely thin double layer and cumulative inductions of HDW inside the double layer produces a narrow zone of HDW immediately at the surface and a broad zone of LDW outside.

32

Figure 16: a, the same charged surface as in Figures 13 and 14; b, the effect of the kosmotropic divalent cation is modified by the presence of the chaotropic Cl- which is needed to to maintain electroneutrality inside the double layer; c, the cumulative effect of these effects, including the doubled excess concentration inside the double layer induces a maximally enhanced HDW in the double layer, together with a large zone of LDW further out.

With Ca2+ as counterion to hydrophobic glass beads, aggregations were

extremely stable, more stable, in fact, than those in the presence of K+ or Cs+, indicating the presence of excess LDW. Beads also floated strongly.

With Tris+ as counterion to DNA, the configuration of water in and outside

the double layer is less extreme. Tris+ is a chaotrope, inducing a configuration like that with K+ as countercation, with the difference that Tris+ has also some kosmotropic properties which moderate the HDW immediately adjacent to the surface.

Chaotropic nonelectrolyes (glucose, urea, trehalose etc.) In bulk water

These molecules, having both chaotropic and kosmotropic moieties partition

into LDW with relatively low partition coefficients. They are highly soluble and induce HDW: they are chaotropes.

At uncharged surfaces They partition into LDW in cellulose acetate films and in polyamide beads,

but induce HDW and diffuse out immediately: i.e. they are not retained or retarded a column of polyamide beads. D-glucose has been used extensively as a probe for LDW in spite of its behaviour as a destroyer of its own preferred environment. Evidence that osmotic pressure gradients are involved in all these experiments comes from the ability of various other solutes which are either excluded from the LDW because they partition into HDW (NaCl, MgCl2, butanol, BzOH) or are too big to penetrate the pores of the polyamide beads (polyethylene glycols). At low concentrations of excluded solutes, glucose induces HDW and is not retarded. As the concentration of excluded solute increases glucose is increasingly retained and the beads swell as water

-

- -

-

-

- -

- +p -p

a b c

Cl-

Cl-

Cl-

Cl-

Ca2+ Ca2+

Ca2+

Ca2+

-

-

- -

Cl-

Cl-

Cl-

Cl-

Ca2+ Ca2+

Ca2+ Ca2+

33

follows the accumulating glucose. At still higher concentrations of excluded solute, the internal volume of the beads becomes constant because water stops moving in. At yet higher concentrations, the beads begin to shrink as the osmotic pressure gradient reverses in sign and glucose is lost again. Maximal retention of glucose occurs when the internal volume is constant. This method has been used extensively in the separation of D- and L-glucose on a polyamide column (Chapter 13)

At charged surfaces They partition into the LDW outside the double layer, reducing its

induction. But, at the same time, they are not strongly excluded from the HDW in the double layer, and decrease the dielectric constant, thinning the double layer and increasing the amount of LDW. These effects are concentration-dependent. Urea, for example, favourite chaotrope of protein chemists, increased the viscosity of dextransulphate solutions at concentrations up to 1M, and thereafter, decreased viscosity. This chaotrope behaved as a kosmotrope at quite high concentrations.

Urea and trehalose both stabilised hydrophobic glass beads at

concentrations up to 2M and thereafter destabilised them. Beads that disintegrated and sank rapidly with Na+, Li+ or H+ as counter ion, became as strongly aggregated as beads with K+, or Cs+ as counter ion in the presence of up to 2 M urea. This is a clear indication that the stabilising activity took place in the double layer, since that was where the kosmotropic cations were destabilising. Not until 5M urea did the aggregations of beads collapse.

These experiments offer an explanation for observations which suggest that

the usual assumption of toxicologists, that the effect of a drug is linearly related to its concentration, need not be valid. Indeed, a harmful effect at high concentrations may convert to a beneficial effect at extremely low concentrations and vice versa. This may go some way to validate the claims of homeopathy.

Kosmotropic nonelectrolytes (betaine, trimethylamine oxide (TMAO),

polyethylene glycols (PEGs), hydrophobic amino acids In bulk solution These molecules, with both chaotropic and kosmotropic moieties partition

into HDW with partition coefficients which range from high (limited by the solubility of the molecule) to low, depending upon the proportions of chaotropic and kosmotropic moieties.

At an uncharged surface They are excluded from LDW in polyamide beads and cellulose acetate, and

promote retention of chaotropes such as glucose. BzOH was used together with glucose to characterise the conditions under which it promoted the retention of glucose. Glucose, alone was not retained on a polyamide column, but glucose and BzOH together were both retained, to a degree. BzOH concentrated in the external solution but also was adsorbed to hydrophobic surfaces inside the pores. This was an

34

example of the operation of the hydrophobic interaction. Adsorption of BzOH depended upon the presence of LDW inside the pore, because both glucose and BzOH were eluted with a strong chaotrope such as KNO3. This is consistent with the proposed mechanism of the hydrophobic effect: it results from the high cost of induction of LDW, and is reversed when LDW is demolished. This must be an important mechanism in enzyme reactions.

At a charged surface Kosmotropes also have competing effects at charged surfaces. They

accumulate in HDW immediately adjacent to the surface, decreasing the dielectric constant, decreasing the thickness of the double layer and increasing the osmotic pressure gradient due to the excess concentration of counterions. At the same time they increase the excess concentration of total solutes in the double layer. These two effects are opposed by the increase in the thickness of the double layer as water follows the kosmotropes into it. The nett effect of kosmotropes, therefore depends on the magnitude of their partitioning into HDW. Butanol, for example increased the fluidity of dextran sulphate solutions because, as a powerful kosmotrope it increased the thickness of the double layer. Betaine and TMAO, on the other hand increased the viscosity of dextransulphate solutions. Their excess concentrations in the double layer are rather slight, and their principal effect seems to be that of decreasing the dielectric constant, thinning the double layer, and, therefore inducing more LDW.

35

Chapter 7

Solution and folding of proteins Natural proteins fold to a configuration in which they are soluble. Most

artificial proteins and many synthetic polymers are insoluble. This means that natural proteins are a highly selected set, so that it is not surprising to find that even these, under abnormal or extreme conditions, form fibre-like insoluble masses. These insoluble fibres form the basis of the protein-folding diseases that occur with age41.

Proteins, like their amino acid components, have either a preponderance of

chaotropic groups or a preponderance of kosmotropic groups. Because of their size (perhaps > 100,000) relative to amino acids (perhaps 111) their interface with water is correspondingly greater. This increases the danger of an excess induction of either HDW or LDW, increased thermodynamic cost of hydration and, finally, insolubility. The balance of HDW and LDW induced on solution of a protein, is, therefore, critical for their survival in solution. The insoluble fibres found in diseases like Alzeimer’s and Type II Diabetes (Dobson, 2002)41 may be a consequence of mutations which upset this balance on proteins normally capable of folding and dissolving. Their synthesis would continue and, because the fibres are not in solution, they would not be eliminated by the usual protective mechanisms. An alternative cause of precipitation of intracellular proteins with age might be that the intracellular composition of solutes and water, which is tightly controlled normally by means of pumps and leaks, becomes compromised, changing the relative concentrations of small chaotropes and kosmotropes and thus the balance of HDW and LDW. This could precipitate some proteins specifically, according to the Hofmeister principle (Chapter 4).

The ensuing damage to a tissue containing such fibrils is then probably due

to the introduction of a surface to which normal proteins can stick and become insoluble. Biological surfaces are, obviously, compatible with the existence of proteins and other molecules in solution. One of the most interesting results of the experiments with glass beads was that the beads (which were not soluble themselves) stuck to any available surface. Thus, in the presence of water, hydrophobic beads (with a large excess of kosmotropic moieties) stuck to plastic (kosmotropic) and glass (chaotropic) surfaces of the containers with great avidity. Hydrophilic beads (with a large excess of chaotropic moieties) also stuck to both plastic and glass. This behaviour was only observed in H2O and D2O, not in ethanol, DMSO, or hexane. The general stickiness increased with the size of the beads from 44 to 600 µm in diameter.

It seemed that this aggregating force was due to the large positive term in the free energy of hydration of the surfaces, increasing with the surface area of contact between beads and water. As in the hydrophobic force (which is one manifestation of this attractive force) there are no specific binding sites involved. A bead adheres to the surfaces merely to decrease the surface area of contact with water and avoid the costly free energy of hydration.

The same stickiness was found when suspension cells of various kinds were

stored in glass or plastic tubes. Red cells, platelets and stem cells all stuck to plastic and glass surfaces and lost viability. Their survival was markedly improved when glass surfaces were treated with dimethyldichlorosilane, which converted the strongly

36

chaotropic Si-OH groups into kosmotropic Si-OCH3. The longest survival, however, was for cells encapsulated in vesicles made from sodium alginate and lysine: i.e a compatible biological surface.

The characteristics of a compatible biological surface are that it is easily

wet; i.e. that it is composed of more-or-less equal numbers of chaotropic and kosmotropic moieties. This corresponds to the extreme solubility of small solutes with both moieties, contrasting with the low solubility of pure chaotropes or pure kosmotropes. The importance of this balance for the solubility of polymers is illustrated by the polyethylene glycols (-O-CH2CH2-)n. These form very soluble high molecular weight polymers (up to 107), whereas both polypropylene glycol (-O-CH2CH2CH2-)n and polymethylene glycol (-O-CH2-)n form insoluble polymers. Presumably (-O-CH2CH2CH2-)n is too kosmotropic and (-O-CH2-)n too chaotropic to be soluble when the surface area of contact with water is too high.

The insoluble fibrils of Alzeimer’s and Type II diabetes are not compatible

in this sense and must act as sticky points of adhesion to other proteins, which, then precipitate out, forming more fibrils.

Folding of proteins

One of the driving forces for protein folding must be the free energy of

hydration of the extensive surface of the unfolded protein; folding is a step on the way toward insolubility. Only native proteins achieve a viable folded state, stopping short of precipitation. Folding and solubility, however, can both be influenced by small solutes. For example TMAO and betaine (two moderate kosmotropes) induce folding of some proteins at relatively low concentrations, but induce precipitation at high concentrations. Urea (a moderate chaotrope) solubilises the same proteins at high concentrations. Many experiments (eg. Yancey et al, 1982) have shown that these two solutes compensate eachother. If TMAO precipitates a protein, urea will reverse that precipitation and restore it to solution. Many organisms contain approximately 2 concentrations of urea for one concentration of betaine or TMAO for optimal homeostasis.

Compensatory mechanisms.

This is an example of compensatory mechanisms which are very common in

physiology. A solute that induces too much LDW is balanced by a solute which produces HDW. Other participants in these compensatory mechanisms are pressure and temperature. For example, Yancey et al42 found that fish which lived in very deep water and, therefore, under high pressures, contained more TMAO (a kosmotrope) than did fish from shallower waters. Again, TMAO induces LDW to balance the effects of pressure which induces HDW.

37

Chapter 8

Synthesis of ATP from ADP plus Pi in LDW and its hydrolysis in HDW

Mechanism and source of energy for synthesis of ATP We showed35 that ATP was synthesised from 1 mM ADP and 5 mM

potassium phosphate in the LDW in dense cellulose acetate membranes, which has already been discussed (Chapter 6). ATP was estimated using either HPLC or the luciferin-luciferase reaction in which ATP causes emission of light. ADP was estimated by HPLC. In the presence of 100 mM NaCl or of 100 mM MgCl2 no ATP was detected. In the presence of 100 mM KCl or of no additions other than the 5 mM potassium phosphate as reactant, ADP was converted quantitatively to ATP. The sum of ADP plus ATP remained constant while ATP increased and ADP decreased over time.

The conclusion was that the synthesis is a spontaneous reaction in LDW, but that the release of chaotropic ATP from the membranes required conversion of LDW to HDW. This was achieved by potassium phosphate and by added KCl, but was opposed by addition of 100 mM NaCl or 100 mM MgCl2, either of which stabilised LDW and prevented release of ATP (see Figure 3).

When ATP is synthesised by the ATPsynthase, a flux of H+ ions is required not, presumably, to provide energy for ATP synthesis, but to break down LDW and allow release of spontaneously synthesised ATP. Similarly, a reverse operation of the, the Na,K-ATPase requires a flux of Na+, and the reverse operation of the Ca-ATPase requires a flux of Ca2+. These cations are all kosmotropes which, at charged surfaces, induce HDW (see Chapter 6).

The energy for ATP synthesis is provided by the high cost of hydrating a surface which strongly induces LDW. If two such surfaces can move together, the LDW is eliminated, but if the structure of the cavity in which the LDW forms is such that the opposing surfaces are held apart, the cost of hydrating the surfaces must be met: LDW forms and is the environment in which synthesis of ATP from ADP and Pi is a spontaneous reaction. The energy is supplied in the form of changes in the free energies of hydration of MgATP, ADP and Pi and in the high cohesiveness of LDW which promotes the removal the water molecule from the transition complex. Thus the free energy change of the reaction

ADP + Pi +Mg2+= MgATP + H2O

becomes negative and the

reaction proceeds spontaneously. When the newly synthesised ATP is released it is stable because it cannot

be hydrolysed by the normal mixture of LDW and HDW. It can be hydrolysed only in HDW, as is discussed next.

38

Hydolysis of ATP, peptides, oligonucleotides, etc.

Water is an extremely weak acid and base. The concentrations of H+and OH- are each 10-7M at 25oC. Dissociation increases but slightly with increasing temperature. This, again, is unusual. Oxyacids of neighbouring elements in the Periodic Table are often very strong; eg. H2SO4, HNO3, HCl, H3PO4. Figure 18: a, a HDW microdomain in liquid water which ionises to produce hydrogen and hydroxyl ions; b, rapid conversion of HDW to LDW to eliminate the osmotic pressure gradients created by ionisation of water in HDW and not in LDW

The H+ ion is the most powerful kosmotrope of all univalent cations; the OH- ion is one of the very few kosmotropes among univalent anions. Water ionises, therefore, in HDW which readily dissolves its ionic products but not in LDW. The immediate consequence is conversion of HDW to LDW to eliminate the steep osmotic pressure gradient created between neighbouring microdomains. Liquid water, therefore, is largely unionised and is a very weak base and a very weak acid. Figure 19: H2SO4 ionises to produce a powerful kosmotrope ( H+) and a powerful chaotrope (HSO4

-). It, therefore ionises in both LDW and in HDW and is a strong acid

The first dissociation of strong acids produces the powerful kosmotrope H+, together with a univalent anion (Cl-, Br-, I-, HSO4

-, H2PO4- or HCO3

-) which is a

OH- H+

H+

H+

H+

OH-

OH-

H+

OH-

HDW LDW

HSO4- H+

H+

H+

H+

H+

HDW LDW

HSO4-

HSO4-

HSO4-

H+

H+

H+

HSO4-

HSO4-

HSO4-

HSO4-

39

powerful chaotrope. Ionisation it thus possible in both LDW and in HDW, and the osmotic imbalance is so slight that little conversion of either LDW into HDW or of HDW into LDW is required.

Figure 20: Protection of ionised water by a chaotrope partitioning selectively into LDW in contact with HDW. It is possible, however, to protect ionised water by means of partitioning of a chaotrope in a contiguous microdomain of LDW, when the osmotic pressure gradient due to ionisation is balanced by an osmotic pressure gradient of opposite sign due to selective partitioning of a chaotrope. We found that DNA with Mg as counterion was hydrolysed in a solution of Tris borate buffer at 53oC. Presumably, the divalent counter ion induced HDW at the surface, surrounding the P-O bonds linking the nucleotides, and the chaotropes Tris and borate partitioned into contiguous LDW, protecting the ionisation of HDW. With Tris as counter cation under otherwise similar conditions, there was no hydrolysis.

This suggests a mechanism for proteases. They often require Ca2+ which, as counterion, induces HDW immediately at the surface. If ionisation of water in HDW is protected by chaotropes, water surrounding the peptide bond to be cleaved is both a strong acid and a strong base. Like all enzyme reactions this has important constraints. There is a narrow range of chaotrope concentrations which can just match the osmotic pressure gradient of ionisation. Too high a concentration demolishes LDW and , consequently HDW. Too low a concentration is ineffective.

H+

H+

H+

H+

HDW LDW

OH-

OH-

OH-

OH-

40

Chapter 9

Mechanism of sodium transport driven by ATP and its hydrolysis

Figure 21: a, the enzyme cavity in its resting state, containing HDW, with Na+ as counter ion to the aspartyl residue that is to be phosphorylated and to two negative sites near the apex; b, MgATP phosphorylates the aspartyl residue, Mg2+, remaining as counter ion. LDW is induced and moves up the cavity, pushing Na+ ahead of it and out the open channel. K+ enters selectively into the LDW; c, the K+ ions balance the osmotic pressure gradient created by ionisation of HDW ,and the phoshate is hydrolysed off. The water reverts to HDW, K+, Mg2+, phosphate and ADP diffuse out and Na+ enters to neutralise the aspartyl residue. Two more Na+ ions enter in exchange for the K+ ions which have left, and the cavity resumes its resting stte in a.

This has been discussed previously17-20 . The new approach is the

introduction of two-state water and the extraordinary power of Mg2+, which has only been apparent since the experiments with glass beads. (This paper has never been published in full). Figure 21 illustrates the steps in this new version of the pump. In the resting state of the enzyme the active site contains HDW. The carboxyl groups of the aspartyl residue (to be phosphorylated) and two others inside the cleft are neutralised by Na+ ions derived from the cytoplasmic side. Although K+ ions are far in excess in the cytoplasm, they are not permitted into the HDW. MgATP enters the cavity, also from the cytoplasmic side, and phosphorylates the aspartyl residue. This is, perhaps, the most crucial factor in the whole reaction sequence. In the cytoplasm, Mg2+ ions are all chelated to ATP, in which two ionised phosphates are close enough together, and appropriately oriented, to neutralise the two positive charges on a single Mg2+ ion, which moves through the solution with the ATP molecule. When, however MgATP phosphorylates the aspartyl residue, the Mg2+ ion is released from chelation and is left to neutralise a single negative charge. That negative charge now belongs to

PO- Mg2+

ADP MgATP

-Na+

- Na+ Na+

_

K+ K+

Na+

K+ K+

Na+Na+

Mg2+ H2PO-

Na+ Na+ Na+

Na+

K+ K+

LDW HDW

a b c

41

the protein and assumes the mobility of the protein. Mg2+, therefore, no long diffuses freely through the solution but is localised to the site of the phosphorylated aspartyl residue. It is in solution, fully hydrated and remains as the much-preferred counter cation. It is preferred above K+ because it is strongly attracted into the HDW in the double layer; it is preferred to Na+ because it has two positive charges and, therefore is held much more closely to and strongly by the negative charge. Most important of all, because it is divalent, it must carry with it into the double layer a univalent anion to maintain local electroneutrality. The cumulative effect of these influences is such that Mg2+ as counter-cation to a chaotropic fixed charge, induces LDW throughout the cavity. This LDW does not instantly appear, but develops over a short time, starting at the phosphorylated site and moving up the cavity.

Na+ ions, including the one that had neutralised the aspartyl residue before

phosphorylation, move ahead of the wave of LDW. The channel through which Na+

and K+ are to exchange is closed because it carries a negative charge and, with K+ as counterion is filled predominantly with LDW. K+ and anion from the extracellular solution cannot enter because the negative charge repels the anion which is the dominant ion, leading into LDW. When, however, the Na+ ions reach the channel, the Na+ ions go preferentially into the small volume of HDW, increasing its volume at the expense of LDW. The channel is now open to Na+ which diffuses out, exchanging for K+ in order to maintain electroneutrality. This exchange illustrates the dominance of the electrostatic attraction of ions over their solvent preference. The two K+ ions become counterions to the carboxyl groups previously neutralised by Na+. The channel is again closed to Na+ and has K+ neutralising its negative charge. K+ in the LDW allows the ionisation of water in HDW the double layer which surrounds and cleaves the O-P bond, releasing a phosphate ion. With hydrolysis of the phosphorylated enzyme, water in the cleft again becomes HDW, K+ inside exchanges with Na+ in the cytoplasm, and the cycle begins again.

In this scheme, phosphorylation by MgATP is required to induce LDW

which drives the Na+ ions out and draws the K+ ions in. Hydrolysis is then required to restore the phosphorylated enzyme to its resting state. The energy for work performance comes from the induction of LDW in the cleft of the phosphorylated enzyme. Its formation requires the energy involved in displacing the water equilibrium. Its highly selective solvent properties, then allow the Na+/K+ exchange to occur spontaneously. Hydrolysis and release of inorganic phosphate follows the exchange of ions. Notice that each stage in this overall procedure depends upon the preceding one: MgATP, being a nett kosmotrope, cannot enter the cavity until it contains HDW, when phosphorylation of the aspartyl group becomes a spontaneous process; the resulting wave of LDW moves Na+ away from the phosphorylation site, opens the channel and allows spontaneous exchange of Na+ and K+; influx of K+ induces ionisation of HDW (see Figure 20) and allows spontaneous hydrolysis of the phosphoryl group; water reverts to HDW, K+ diffuses out of the cavity and Na+ re-occupies the carboxyl sites. The role of ATP is to phosphorylate the enzyme and to introduce Mg2+ into the cavity, so that it becomes the preferred counter cation.

42

Chapter 10

Ion channels

Figure 22: a, the entrance compartment of an ion channel containing LDW and, therefore, closed to Na+ and Ca2+; b, K+ and an anion partition into it selectively; c, the resulting osmotic presure gradient is abolished by conversion of the water to HDW, into which Na+ and Ca2+ diffuse while K+ and anion, having destroyed their preferred environment diffuse out; d, LDW reforms, because that was the state of the water in the absence of solutes, the compartment is again closed to Na+ and Ca2+.

In the discussion on surfaces it was shown that, depending on the local

surface moieties, clefts or pores in proteins can be enriched in either LDW or HDW. If entrance compartments of ion channels are enriched in LDW, they are effectively closed to kosmotropic ions such as Na+ and Ca2+, but open to K+ and univalent anions. See Figure 22. Selective accumulation of K+ and anion into LDW, then converts LDW into HDW. In this state the channel is open to kosmotropic ions. If the filter region is specific for Na+, then Na+ diffuses spontaneously into the cell, followed by an anion through an anion channel to maintain electroneutrality. K+ and anion, which opened the channel for Na+, diffuse out into the extracellular solution, having destroyed the LDW which attracted them in. If the driving force for transmembrane K+ movement is inwardly directed and the filter specific to K+, this might also be a K+

channel. Since univalent anions have greater affinity for LDW than has K+, they lead the inward diffusion that opens the channels. Experiments have shown, as expected, that electroneutrality must be conserved in the entrance compartment. Many channels are voltage-gated: i.e. they open in response to induction of a more positive membrane potential. Since the anion leads diffusion of a chaotropic ion pair into LDW, too negative a potential would prevent channel opening. With a more positive potential the anion would be attracted in both by its highly favourable partitioning into LDW and by the lowered membrane potential. The cation would follow.

Evidence that chaotropic ions open channels

Physiologically, intracellular concentrations of Na+ and K+ are controlled by

a steady-state balance between the rates of active Na+ efflux and K+ influx through the Na,K-ATPase and rates of passive Na+ influx and K+ efflux through selective channels. The Na,K-ATPase is inhibited specifically by ouabain, by depletion of

Ca2+

filter Na+

a b

A-

K+

Ca2+

filter Na

+

K+

A- Ca2+

filter Na

+

c d

a b

A-

K+

Ca2+

filter Na

+

K+

A- Ca2+

filter Na

+

43

intracellular ATP and by absence of either intracellular Na+ or extracellular K+ (refs). When cells and tissues are treated with ouabain or anoxia, they lose intracellular K+ and take up NaCl and water. With time this swelling becomes irreversible and cells die. Presumably, the passive channels are still opening and closing, but there is no active efflux of Na+ or influx of K+. When, however, the same cells are treated with a K+-free medium, cells do not swell and do not take up NaCl. It is known, from experiments with isolated membranes that, under these conditions, active influx of K+ and efflux of Na+ do not take place. That cells fail to swell and take up NaCl must, therefore, mean that, in the absence of K+, passive channels are also inhibited.

Ca2+- activated K+ channels Figure 23: a, the empty entrance compartment of a calcium-activated potassium channel containing HDW, excluding K+; b, Ca2+ enters, selectively, with anion; c, the kosmotropic Ca2+ converts HDW to LDW, and Ca2+ and anion diffuse out as K+ enters and continues through the K+- selective filter region; d, the empty compartment re-introduces HDW.

filter

a b

filter

K+

A-

filter

c d

a b

filter

Ca2+ filter

K+

K+

Ca2+

K+

44

Chapter 11

Preservative solutions We have used this concept of channel-opening to design solutions in which

cells achieve a state of dormancy, and can survive without energy input for days or weeks44.

Ideally, solutions contained no ionic chaotropes. Chaotropes which were permitted, however, were of large size (eg. Raffinose, MW > 500) so that they were excluded from channel entrance compartments by size. Other solutes, making up the osmolality of the solutions to 290-300 mOsM, included betaine, trimethyl amine oxide (TMAO), Na2SO4 and trisodium citrate. Solutions also contained 1.75 mM CaCl2, which was necessary to maintain the integrity of the plasma membrane. In vivo the phosphate headgroups of membrane lipids prefer Ca2+ as countercation because the divalent cation has high affinity for the HDW surrounding them and strongly induces LDW which, in turn, promotes assembly of phospholipids into bilayers. In the absence of Ca2+ and K+, Na+ is the only available countercation. The consequence is a weakening of the water-driven normal packing of the membrane which is necessary for maintaining its barrier properties (Chapter 6; compare the induction of LDW when Na+ is countercation with that when Ca2+ is counter-cation).

The simplest solution contained only NaCl and TMAO and CaCl2. This was permissible, despite the chaotropic Cl-, because the important principle of conservation of electroneutrality excluded Cl- from the channel when the only cation present was Na+: the potency of Na+ as a kosmotrope is greater than that of Cl- as a chaotrope. Solutions were unbuffered; the pH was approximately 8.

Although it was simple to exclude ionic chaotropes from preservative solutions, endogenous chaotropes often carried over from the physiological media in which the cells originated. At pH 7, phosphate was half chaotropic H2PO4

- and half kosmotropic HPO4

2- , while the bicarbonate/CO2 buffer was half chaotropic HCO3-.

Together with residual K+, these chaotropic anions were in high enough concentrations to continue opening channels. These experiments have highlighted the extreme potency of low concentrations of ionic chaotropes, which seriously impeded preservation at pH 7. At pH 8, however, when the residual endogenous concentrations of H2PO4

- and HCO3- were drastically reduced, preservation improved. This problem

was solved by a preliminary washing in a solution at pH 8 to remove effectively K+ and Cl- as well as H2PO4

- and HCO3-.

Suspension cells such as blood cells were most difficult to preserve in liquid storage, because their isolation required centrifugation. Centrifugation acts as a pressure on the settling cells, and converts the LDW which maintains ion channels in their closed conformation, into HDW, allowing inward fluxes of Na+, Ca2+, Cl- and water and efflux of K+. Both gain of Na+ and loss of K+ impaired the desired state of dormancy and limited the time for which cells could be held in a viable state. The excess concentration of intracellular Na+ increased residual metabolic rate by inducing HDW at charged interfaces and decreasing the nett viscosity of the intracellular compartments, so that diffusion was rapid and enzymes able to undergo the conformational changes which are a feature of their activity. Loss of K+ was equally deleterious because the concentration of channel-opening ions in the

45

extracellular medium increased. Red cells, for example, survived without haemolysis for only 6 days when they were derived by centrifugation of whole blood. This storage time lengthened to more than 13 weeks when cells were protected against centrifugation damage by a preliminary washing with isotonic sodium acetate. Whole blood was diluted with excess sodium acetate before centrifugation. This kosmotropic salt was excluded from the entrance compartments of channels and stabilised LDW so that it resisted the increased pressure due to centrifugation. Channels remained closed and cells neither lost K+ nor gained Na+. This washing procedure was repeated because it also had the effect of diluting out endogenous K+ carried over from the plasma.

Effects of temperature on storage Liquid storage was usually at 4oC, because of the assumption (which, later, was realised to be erroneous) that at low temperatures residual metabolism would be minimised and the membrane most stable. Again, however, there were paradoxical effects of an increase in viable storage time with increase in temperature up to 38oC. As Robinson and coworkers6 showed, the fraction of HDW in the pure liquid increases with increasing temperature. If entrance compartments of ion channels are, indeed filled with LDW, other things being equal, an increase in HDW must increase the enhancement of LDW. This follows because the cumulative effects of chaotropic surfaces, pressure gradients due to surface roughness and the small diameter of the cavity are now compounded by an increase in HDW of the hydrating water. Thus, while, an increase in temperature, increased HDW in bulk water, it increased LDW in ion channel entrance compartments. Channels, therefore, were more difficult to open, spent more time in the closed configuration and cells remained viable for longer periods. We found, for example, that solutions containing NaCl or sodium gluconate as the ionic component of the storage solution increased storage to 6 weeks at 21oC compared with 6 days at 4oC. The nature of the anion proved to be crucial for this preservation of cells at elevated temperatures. For example when the anion was citrate, red cells which survived for >13 weeks at 4oC, survived for only four days at 21oC. Similarly sulphate was better at 4oC than at 21oC. Ionisation of weak acids increases with increasing temperature, so that the chelating anions, which form rather insoluble salts with Ca2+ , were at higher concentrations at 21oC than at 4oC, and complexed Ca2+. The only other cation present, Na+, then became counterion to the phospholipid headgroups of the membranes. The main driving force in folding membranes is that of avoidance of formation of LDW, with its high thermodynamic cost. As with the coated glass beads, Na+ as countercation decreased the amount of LDW induced at kosmotropic surfaces, and destabilised the aggregated form. The failure of storage in these solutions at 21oC was due to leaky membranes. Sodium gluconate also ionises more at increased temperatures, but this was beneficial to storage, because the hydrated gluconate ion together with a sodium ion was too large to enter the entrance compartment of the channel. The neutral gluconic acid, on the other hand, was small enough to enter and is, itself a chaotrope. Morevoer, as the gluconate anion is a chaotrope it does not precipitate calcium. Therefore increased ionisation of sodium gluconate is beneficial.

46

HCl is fully ionised so that its dissociation is little effected by increased temperature. It was equally good at 4oC and at 21oC.

Freezing of cells When unprotected cells are frozen they do not survive thawing. It is generally thought that their death is a consequence of damage by ice crystals. In terms of two state water, there is another, probably more important source of damage. As the temperature decreases, channels with their entrance compartments enriched in LDW become more permeable and stay longer in the open configuration. Moreover, as ice crystals begin to form, the concentration of extracellular Na+ increases. For these two reasons, there is a rapid influx of NaCl and efflux of K+. As Na+ replaces K+ as counterion to proteins, the viscosity of the cytoplasm decreases, higher-order structures which normally exist among proteins disassemble; the resting state of the cell no longer exists. Residual metabolism accelerates in the fluid cytoplasm in which enzymes can more readily undergo the conformational changes necessary for their function. The Na,K-ATPase, an avid consumer of ATP, is stimulated by the increase in intracellular Na+. When the cell is thawed, it rapidly uses up any residual energy stores before it can take up more glucose, uptake of which is now impaired because of the loss of a sodium gradient. Cells die. Small molecules which can cross the membrane spontaneously (dimethyl sulphoxide, glycerol, etc.) protect cells against this damage, not by preventing the influx of Na+

but by compensating for its effects by protecting intracellular LDW. NaCl has three deleterious effects on residual metabolism: Na+ replaces some K+ as counter ion, and NaCl increases the thickness of the double layer, both promoting HDW, and, specifically, intracellular Na+ stimulates the Na,K-ATPase, depleting ATP stores rapidly. High concentrations of the small cryoprotectants are needed to shrink the double layer enough to restore it to its pre-Na+ level. DMSO and glycerol are both used at concentrations of 1-2 M.

Freezing in solutions devoid of ionic chaotropes The alternative method of protecting cells against this mode of freezing damage, is to keep channels closed during freezing so that there is no influx of NaCl and water and no efflux of K+ We have used this method for freezing stem cells, platelets and jurkat cell lines with good recovery of cells . In the controlled freezing method using DMSO or glycerol, cooling and warming are generally rather slow. In the method using solutions devoid of chaotropes freezing must be rapid. We immersed cells without any insulation at either –196oC or –140oC, and thawed them rapidly at 38oC. We also rapidly froze and then lyophilised platelets and plant cells in culture. The need for rapid freezing and thawing probably arises because of the peculiarities of two-state water at its freezing point and below.

47

Two-state water at 0oC. Water has long been known to have anomalously high melting and boiling points ( Table 1). Its analogues in Group 6 of the Periodic Table (H2S, H2Se and H2Te) behave as one would expect for normal liquids: as the molecular weight increases, melting and boiling points increase, because it takes more energy to melt or vaporise the larger molecules. If one plots melting points and boiling points against molecular weight and extrapolates to molecular weight 18 (for water) one finds that H2O, if it followed the pattern established by the other three compounds, should melt at about –90oC and boil at about –75oC. The observed melting point of 0oC and boiling point of 100oC have been attributed to the fact that H2O molecules are mutually hydrogen bonded, whereas molecules of H2S, H2Se and H2Te are not. This, then, accounts for the extra energy required to melt and vaporize water: hydrogen bonds, holding molecules together in the crystal, and residual hydrogen-bonds in the liquid that must be broken. If, however, there are two populations of water molecules with very different hydrogen-bond strengths, freezing behaviour is more complex. LDW, with its strong hydrogen bonds should freeze at the highest temperature; i.e. at 0oC. As it freezes, the residual liquid becomes enriched in HDW, which has a lower freezing point. There is, however, a limit to the extent to which the equilibrium between HDW and LDW in the liquid can be displaced. At each temperature including and below 0oC, residual water in equilibrium with ice must be largely HDW. Since this water cannot spontaneously convert to LDW, because of the thermodynamic cost of extreme displacement of the LDW/HDW equilibrium at constant temperature and pressure, the barrier to its ionisation is lifted, and it is extremely reactive with higher-than-normal concentrations of both H+ and OH-. Therefore the time that cells spend in contact with this water must be minimised. We have found that cells progressively die at –80oC and die very rapidly at –20oC. We have shown that hydrolysis of p-nitrophenol still occurs, although slowly at –80oC. The need for rapid freezing also applies to lyophilisation.

Sodium as a key player rather than a messenger.

Sodium and calcium which enter cells in response to receptor-ligand interactions are usually called second messengers. A case can be made that they are not just messengers, which are in no way responsible for consequences of their messages, but key players in a sequence of events that they initiate. Resting cells In a resting state, the principle intracellular cation is K+. Much or even most of this K+ is site-localised as countercation to carboxyl groups on proteins. This has been estimated by adding the number of exposed carboxyl groups on representative proteins and comparing that number with the known intracellular K+ content. This was very approximate, but the answer was that they are of the same order. Cytoplasmic Ca2+ is very low (<10-7 M) and Na+ is of the order of 5-10 mM. The imbalance of Na+ and K+ is maintained by activity of the Na,K-ATPase; the low cytoplasmic concentration of Ca2+ by Ca-ATPase on the plasma membrane and/or a Na+/Ca2+ exchanger, together with intracellular Ca-ATPases on endoplasmic reticulum etc. Mg2+ is largely complexed to ATP and other phosphates.

48

Intracellular proteins are folded in such a way that about half the hydrophobic amino acid residues are out of contact with water but most charged groups, positive and negative are in contact with water. This means that the effects of these cations (and other solutes) will be principally their effects on the properties of water in and associated with double layers, in which the fixed charges (-COO- and –NH3

+) are, themselves, chaotropes. With K+ as counter-cation, LDW is very much enhanced (as illustrated in Fig.13). The consequence is aggregation of proteins into higher-order structures, with residual water still very much enhanced in LDW. Diffusion is slow and enzyme activity inhibited by the increased cytoplasmic viscosity which prevents the conformational changes of enzymes which are an integral feature of their activity. Metabolic rate is low. Influx of Na+ converts LDW to HDW, loosening the aggregated structures (Figure 14), facilitating diffusion and conformational changes and allowing activities such as sliding of filaments (muscle cells) and transcellular transport (epithelial cells) to take place. While in simple solutions the differences between Na+ and K+ appear slight, they are amplified several times at charged surfaces, and inside cells which are concentrated solutions or gels of polyelectrolytes.

49

Chapter 12

Formation of bone Bone consists principally or entirely of Ca3(PO4)2 and collagen. This, in itself is surprising because, in blood, the concentration of Ca2+ is only 2.5 mM, and that of PO4

3- at pH 7.4 is about micromolar. How, then does Ca3(PO4)2 ever reach a high enough concentration to precipitate out ? If we believe the published solubility product of Ca3(PO4)2, it should, and normally does, remain in solution at neutral or high pH. As bony spikes do not appear randomly in the blood, which everywhere has a pH of 7.4, some special set of conditions must be associated with formation of bone where it is wanted.

Phosphates

The phosphate species have many very important properties in two-state water, which give pH a new dimension. Of the three: PO4

3- partitions very strongly into HDW and strongly induces LDW; HPO4

2- partitions less strongly into HDW and induces LDW less strongly; H2PO4

- partitions very strongly into LDW and induces HDW. These three species are intimately involved in synthesis and dissolution of bone. Calcium Ca2+ partitions very strongly into HDW and induces LDW. A general rule is that the more highly charged is an ion the more strongly it partitions into HDW and induces LDW. In order that bone may form in blood, there must be sequential steps: (i) Ca2+ and PO4

3- must locally concentrate in existing bone (ii) The concentrated Ca3(PO4)2 must crystallise on the surface of existing bone (iii) There must be a mechanism to prevent continuing proliferation of bone

i) Concentration of Ca2+ and PO43-

The surface of bone, with Ca2+ ions countercations to phosphates is highly

likely to have the configuration shown in Figure 24 (compare Figure 16). Figure 24: the surface of bone, with HDW immediately adjacent to its surface and LDW further out. (see Chapter 6)

bone

HDW LDW

50

Since the surrounding medium contains K+ and anionic chaotropes, the LDW is not constantly present, but forms, collapses and reforms. Thus the HDW is accessible to solutes which partition preferentially into it. These solutes are Na+, Ca2+, H+ and HPO4

2-. PO43- is not included because its concentration is too low for it

to be a significant player. As, however, HPO42- accumulates into HDW, it is rapidly

converted from a modest kosmotrope to a potent kosmotrope, according to Figure 25. Figure 25: rapid conversion of HPO4

2- to PO43- plus H+ in HDW.

As the concentrations of highly selected ions increases, water follows from the contiguous zone of LDW, increasing the volume of HDW. In this way, both the concentrations and the amounts of the species of interest (Ca2+ and PO4

3-) increase locally. While HDW is increasing, neighbouring LDW is decreasing, as kosmotropes in HDW increase more rapidly than chaotropes in LDW. The contiguous zones of HDW and LDW now appear as in Figure 26. Figure 26: relative sizes of HDW and LDW after accumulation of kosmotropes into HDW.

ii) Crystallisation of Ca3(PO4)2 After a critical time the excess concentration of kosmotropes in HDW is

balanced by the excess concentration of chaotropes in the small residual volume of LDW. Flux of water stops and further uptake of kosmotropes into HDW creates an osmotic pressure gradient which is eliminated by conversion of HDW to LDW. This is similar to the conversion of HDW to LDW in calcium-activated potssium channels (Chapter 10). Na+, H+, HPO4

2- diffuse out of the LDW, but Ca3(PO4)2 which now exceeds its solubility product in LDW, crystallises. This is illustrated in Figure 27.

HPO42- PO4

3- + H+

bone

HDW LDW

51

Figure 27: Crystallisation of Ca3(PO4)2 from LDW in which it is highly insoluble.

The efficacy of this method of growth of bone is controlled by the flexibility of the cavity containing HDW. A rigid cavity cannot swell, so that movement of water is zero and the amount of Ca3(PO4)2 accumulated before HDW collapses is negligible. A flexible cavity, on the other hand, allows influx of water and kosmotropes until the limit of its extensibility is reached. Cells which orchestrate this mechaism at their surfaces are osteoblasts.

iii) Regulation of controlled growth of bone.

The continuous production of bone by osteoblasts is matched by a continuos

dissolution of bone by osteoclasts, so that a steady state can be reached.This is similar to the steady state of intracellular Na+ which is maintained by means of approximately equal numbers of pumps which eliminate it and channels that allow it to diffuse spontaneously in. . The plasma membranes of osteoblasts carry proton pumps which acidify the adjacent bone. This converts PO4 3- into the highly soluble H2PO4

-, dissolving the bone locally. The steady state of bone is modified when net growth is needed, sustained when growth is no longer needed but, like many steady states, declines with age.

bone HDW LDW

Ca3(PO4)3

Na+

H+

HPO42-

52

Chapter 13

Origin of chirality

The remarkable solvent properties of high and low density waters suggested (as a remote possibility) that, somehow, they might be responsible for the advent of the extreme bias that has always existed in biochemistry in favour of L-amino acids and D-sugars. If this seems to be stretching credulity further than is generally thought acceptable, it must be remembered that no other plausible mechanism has been suggested, and that there are already many surprises for conventional wisdom in attempts to elucidate properties of water at surfaces. Predictably, funding for this project was not available. Experiments, therefore, were confined to weekends and evenings, with the additional advantage that they did not excite the expected premature comments and criticisms from colleagues for engaging in such a mad pursuit. The first experiments with D- and L- glucose were sufficiently positive to encourage what turned out to be a long and painful series of often unreproducible experiments. Persistence was made possible by two things. First, there had been enough indication that there was a significant difference in the partitioning of these two enantiomers between high and low density water, to feel confident that, if the right conditions were met, reproducibility would follow. Secondly, so many experiments with other solutes had shown that the properties of water at surfaces oscillated with any number of variables (time, concentrations of chaotropes and kosmotropes, temperature, pressure) that it should merely be a matter of time before all these were under control. It took a long time. Drost-Hansen45 has shown in many experiments that water at surfaces does not change momotonically with temperature. There are maxima and minima in water-related properties. In developing the methods for a patent on separation of isomers (US663860 patent), I used the experimental result shown in Figure 28.

Figure 28: the effect of increasing concentration of ethanol on the pH external to microporous polyamide beads soaked for 24 hours in a solution containing 5 mMKPi (pH7). In this extremely simple experiment, beads were soaked in a series of solutions containing a constant concentration of potassium phosphate and variable concentrations of ethanol. The intention was to find the concentration of the kosmotrope ethanol which would just balance osmotically the KH2PO4 that partitioned selectively into LDW in the pores of the beads. In the absence of ethanol

0.1 1.0 10.0 100.05.0

5.2

5.4

5.6

5.8

6.0

6.2

6.4

6.6

6.8

7.0

[Ethanol] (mM)

pH

53

KH2PO4 accumulated in LDW and rapidly demolished it, so that the pH of the external solution was 7, the same as that of the original solution. As ethanol increased from 0 to 5 mM, external pH decreased, slowly at first and then rapidly to 5.3, a decrease in pH that would be rated worthy of a proton pump. In this range of concentrations, the kosmotropic ethanol, largely restricted to the external solution, increasingly compensated for the increasing internal concentration of KH2PO4, which characteristically caused swelling of the beads as water followed the accumulating KH2PO4. At the same time the kosmotropic ethanol induced LDW in the external compartment. In the external solution loss of KH2PO4 left an excess of kosmotropic HPO4

2- and H+, decreasing the external pH, and helping ethanol to induce LDW . Water movement slowed down as the excess concentration of internal KH2PO4 over external ethanol decreased. When ethanol was 5 mM the inward flux of water stopped. Similar changes in volume were shown in Figure 12. Following this halt in water movement, further increments of ethanol merely increased LDW in the external solution, so that there was no longer specific accumulation of KH2PO4 into the pores: external water was becoming equally enhanced in LDW.

Whatever the mechanism of these changes may be it is clear from an inspection of Figure 28 that, in order to separate solutes which partitioned asymmetrically between HDW and LDW, it was first necessary to identify the concentration of kosmotropes, which exactly balanced the given concentration of chaotrope, so that the conditions of the experiment were such that the difference in solvent properties of the two compartments was maximised.

This explained many of the failed experiments: if the concentration of kosmotrope used to balance the accumulated chaotrope was too far outside a narrow range of concentrations, internal and external water were essentially the same. Specificity was lost. This critical concentration is illustrated in Figure 31.

Figure 29: Separation of D- and L-glucose in 1 g of washed P-6 gel mixed for 18 hours in 7.5 ml solutions containing 5 mM tetramethyl ammonium chloride, 10 mM D-glucose, 10 mM L-glucose, 3 mM azide and graded concentrations of ethanol. Gel was mixed with solution and allowed to stand for the time specified. Internal volume was estimated by the height of the gel which increased with swelling. D-glucose was measured in the supernatant using an Esprit glucometer which did not measure L-glucose. L-glucose was labelled with 14C and estimated by counting. Azide was added to ensure that D-glucose was not metabolised by microorganisms

0 10 20 30 406.6

7.0

7.4

7.8

8.2

8.6

9.0

9.4

[D-glu]5

7

9

11

13

hgt of gel[L-glu]

[EtOH]

heig

ht o

f gel

[glucose] (mM

54

during the 18 hours. The greatest separation between L- and D- glucose occurred in the plateau of internal water content. There was little separation either before or after this critical range. Ethanol, of course is an extremely weak kosmotrope and better separations were obtained with benzyl alcohol, as shown in Figure 32.

Figure 30: Separation of D- and L- glucose in a P-6 gel after 18 hours of mixing in a solution containing 25 mM tetramethyl ammonium chloride, 3 mM sodium azide, 20 mM D-glucose and 20 mM L-glucose, and graded concentrations of benzyl alcohol.

Figure 31 illustrates another pitfall in these experiments.

Figure 31: the experiment of Figure 30 allowed to stand for 4 days. After 4 days the extreme swelling of the gel beads caused by the powerful

chaotrope tetramethyl ammonium chloride reached the limit of the extensibility of the gel pores; its further uptake, therefore, converted LDW to HDW, abolishing internal LDW. This had been observed in experiments with the chaotrope alone. Swelling increased up to 4 days, when beads collapsed back to their initial size.

The power of this chaotrope arises because of its size. This underlines the essential criterion for a chaotrope (Chapter 6). The tetramethyl ammonium has a considerable hydrophobic character which, alone, might be expected to induce LDW. Superimposed on this hydrophobicity, however, is the negative charge distributed over the whole ion. This converts it into a chaotrope and, is clearly dominant. The

0 5 10 15 20 2515

20

25

30

[D-glu][L-glu}

[BzOH] (mM)

[glu

] e (m

M)

0 10 2015

20

25

30

[D-glu][L-]

[BzOH] (mM)

[glu

] e (m

M)

55

whole ion partitions into LDW. As discussed in Chapter 5 the partitioning of an ion or molecule is determined by the cumulative effect of three contributions to its free energy of hydration. Since none of these is known individually, the partitioning is a matter for experiment to decide.

Rapid column experiments

Most experiments designed merely to demonstrate a difference in partitioning

between enantiomers were simple column elutions. Figure 34 shows a reproducible result with D- and L- glucose, labelled with 14C. Notice that this is not called a typical result because many more such experiments gave negative than positive results.

Figure 32: Result of elution of D- and L- glucose from a P-6 column. 1 g of dry gel was pre-treated with 0.2 M n-BuOH for 24 h, 1 ml sample (40 mM DL-glucose, 0.2 M BuOH) was put on and eluted with 0.2 M BuOH.

When DL-glucose was layered on a column prepared in water and eluted

with water, there was no retention at all of D- or L-. D-glucose is a chaotrope and, apparently a stronger chaotrope than is L-glucose a kosmotrope. BuOH, in this experiment was used as a kosmotrope to balance the accumulation of D-glucose in LDW.

Figure 33: Elution of D- and L- lysine from a P-6 column. 1 g P-6 gel was pre-treated with 50 mM n-BuOH for 21h; 1 ml of 20 mM L-lysine, 20 mM L-lysine, 40 mM n-BuOH containing 14C-L-lysine was put on the column and eluted with 15 ml 50 mM n-BuOH, followed by 20 mM NH4HCO3. Total lysine was determined from its absorbance relative to that of 50 mM BuOH at 210 nm, using a standard curve of lysine in 50 mM BuOH, and diluting samples where necessary, 50 mM BuOH. L-lysine was determined by scintillation counting and D-lysine by difference. Following

0 10 20 30 40 500

1

2

3D-glucoseL-glucose

Volume (ml)

[glu

cose

](mM

)

0 10 20 30 400

1

2

3

4

D-lysineL-lysine20 mM NH4HCO3

Volume (ml)

[lysi

ne] (

mM

)

56

addition of NH4HCO3 total lysine could not be determined. This pattern of elution was reproducible on the same batch of gel. Figure 34: Elution of D- and L- lysine from a different batch of P-6. The method was the same as for Figure 35 except that the concentrations of D- and L- lysine were 10 mM and the second eluting solution was 50 mM KH2PO4. By making corrections for (i) the effects of changing solution composition on the absorbance of lysine at 210 nm and (ii) NH3 eluted from the gel, total lysine was measured up to 30 ml. This was extremely reproducible on this batch of gel. Both D- and L- lysine were totally recovered. These two experiments with DL lysine show that differences and similarities exist between experiments with changing gel batch, changing concentrations of enantiomers and changing eluting chaotropes, but the patterns of elution were essentially the same. The greatest problem which had to be faced was that enantiomeric biomolecules were always, themselves either chaotropes which tended to destroy LDW or kosmotropes which tended to destroy HDW. For this reason more poweful chaotropes (eg tetramethyl ammonium ion) and kosmotropes (eg butanol or benzyl alcohol) were used to dominate stabilisation of the two zones of water so that the enantiomers could partition without abolishing the conditions that were set up to separate them. Figure 35:Elution of K+ D-glutamate and K+ L-glutamate from Dowex 50W x 16 in its 0sodium form. L-glutamate was labelled with 14C, DL-glutamate estimated by absorption at 210 nm and D-glutamate obtained by difference.

0 10 20 300

1

2 L-lysineD-lysine

50 mM KH2PO4

Volume (ml)

[lysi

ne] (

mM

)

0 10 20 300.0

0.5

1.0

1.5L-glutamateD-glutamate

Volume (ml)

[glu

tam

ate]

(mM

)

0.1 M MgCl2

57

Figure 36: Elution of D- and L- glucose from a Dowex anion exchange column in its chloride form. Retarded D-glucose was eluted with 50 mM MgCl2. Ion exchange resins were most powerful in separating enantiomers, presumably because their zones of HDW and LDW were most stable. Note that a kosmotrope (MgCl2) is needed to elute the chaotropic L-glutamate from a cation-exchange column and the chaotropic D-glucose from an anion-exchange column.. Another indirect methods of demonstrating partitioning of the biologically active enantiomers is illustrated in Figure 37 glutamic acid aspartic acid glucose L- D- L- D- L- D- L- D- L- D- + PEG -PEG + PEG +PEG -PEG

Figure 36: Polyamide beads (P-6) mixed with 10 mM amino acid or glucose, with and without 10% PEG 20 M. In the absence of polyethylene glycol, L- amino acids and D-glucose partitioned selectively into the LDW inside the beads, causing swelling of the beads. In the presence of PEG which was too big to enter the gel pores, beads shrank because of the osmotic pressure gradient created by the excluded PEG. PEG has been shown to induce LDW extremely strongly. Thus L-amino acids and D-glucose partitioned

00.10.20.30.40.50.60.70.80.9

1

0 5 10 15 20 25 30

Volume (ml)

[glu

cose

] (m

M)

D-glucose L-glucose

58

preferably into the external solution and the resulting swelling opposed the PEG-induced shrinkage.

Factors that affect reproducibility

Attempts to separate any solutes which partition differently between LDW and HDW are inevitably complicated by the tendency of each solute to demolish the very type of water that it prefers. This would not have been a problem with the original separation at clay surfaces because concentrations of both enantiomers were low and equal, so that they would compensate eachother osmotically.

For the record, however, a list of factors demanding attention in laboratory experiments follows:

i) A combination of powerful chaotrope (eg. tetramethylammonium

chloride) and powerful kosmotrope (eg. benzyl alcohol or butanol) can together determine the distributions of LDW and HDW so that the relatively weak enantiomers have little effect.

ii) The time of contact between gel and solution is crucial because the system will oscillate with time (see Figures 30 and 31).

iii) Temperature and pressure must be constant iv) The relative amounts of gel and of solution must be constant. v) The quality of water used in these experiments and for washing the gel

must be constant. The experiment illustrated in Figure 28 used distilled, deionised water. When it was repeated with MilliQ water the minimum in pH shifted from 5mM to 25 mM ethanol. Presumably the MilliQ water was of greater purity and retained fewer non-ionic kosmotropes which powerfully modify the properties of water even at trace concentrations. The experiment of Figure 36 was obtained using distilled, deionised water and could not be reproduced using MilliQ water.

vi) HDW exists at the immediate surfaces of both polyamide and ion-exchange gels. With repeated use, therefore, end amide groups in the polyamide and charged groups in the charged gels hydrolyse, changing the fundamental properties of the gels.

These problems, exasperating in the laboratory, did not apply to the primordial separation which had a leeway of millions of years, not acceptable to granting bodies.

Origin of chirality Part 2

It is not enough that L- amino acids and D-sugars partition into LDW: this does not account for their inclusion exclusively into biopolymers. Suppose, though, that the enantiomers of lysine have separated into L-lysine in LDW and D-lysine in HDW. The synthetic reaction producing polylysine can only happen in LDW (see Chapter 8). Therefore there is immediately a strong bias in favour of L-peptide over

59

D-peptide. This bias is perpetuated as L-peptides are retained in LDW while the lower concentrations of D-peptides accumulate in HDW where they hydrolyse. The condensation of L-amino acids and D-sugars into polymers in LDW progressively decreases the total concentration of solutes, thus protecting LDW against conversion to HDW. This can be very simply demonstrated by incubating amino acids with a small-pored silica gel which has much in common with primordial clays. The Si-OH and S-O- groups are both chaotropes. This, together with the porous structure of the silica gel induced stable populations of LDW. In one experiment, 5 mM lysine and 5 mM glycine were mixed with silica gel and incubated at 38oC in the presence of Biuret reagent which dyes peptide bonds blue. The higher temperature was chosen to stabilise the LDW associated with the surface. Lysine was D- or L- or DL. Because there was no extraneous solute to release the product from LDW, the gel itself stained blue, with no peptides released into the supernatant. The blue colour began to appear after one day. When there were no amino acids present or no silica gel, there was no blue stain; with D-lysine the blue stain was faint; with both L- and DL-lysine the blue stain was intense. Because of their different partitioning between LDW and HDW, separation was better from DL lysine than from L-lysine alone; i.e. D-lysine helped to protect LDW from destruction by L-lysine.

H2O D- L- DL- Figure 37: development of the blue dye biuret with formation of peptide bonds in the presence of no amino acids, D-lysine, L-lysine and DL-lysine. By eye, there was no difference between L- lysine and DL-lysine, presumably, because D-lysine, to a degree, balanced the osmotic pressure gradient created by L-lysine, and prevented conversion of LDW into HDW. We also showed polymerisation of D-glucose in LDW. Strips of cellulose acetate containing LDW (see Chapter 3) were soaked in solutions containing either D- or L- glucose. HPLC reported high molecular weight fractions of D-glucose but not of L-glucose. In this experiment glucose, itself, was the chaotrope which broke down LDW and released the synthesised polymer. An important feature of this mechanism of selection is that it is not random: life on another planet would also consist of L-peptides and D-sugars. So when the Martians land we can confidently greet them with extended right hands.

60

Chapter 14

Coupled reactions in Biochemistry

The original formulation of the concept of energy transduction was stated in terms of coupled reactions. One reaction consisted of the work performance and had a positive free energy change. The second reaction was involved in the overall unitary process which performed the work function, but had a negative free energy change. To take a specific example: the Ca-ATPase transports Ca2+ ions from a concentration of, perhaps, 10-6M in the cytoplasm into an external solution with a concentration of perhaps 10-3M. This is the work performance and has a free energy change given by : ∆G = RTln10-3/10-6 = RTln103 kJmole-1 This is obviously a rather large positive quantity. The second participating reaction is the hydrolysis of ATP, which has the rather larger negative free energy change of approximately –30 kJmole-1 Since, free energy changes of individual reactions participating in a single overall process are additive, the unitary process of transport of Ca2+ against a concentration gradient has a free energy change given by: ∆Gtrans = (RTln103 – 30) kJmole-1 Without participation of the hydrolysis of ATP, the reaction has a positive free energy change and will not take place. Together, however, if the sum of the individual free energy changes is negative, the reaction will take place. Since the reaction does take place we can assume that the calculated ∆Gtrans is, in fact, negative. Notice that it does not matter what happens in detail inside the enzyme active site: whatever the molecular mechanisms by which ATP hydrolysis enables the combined reactions to happen, they will happen. If the biochemists on the block had realised that they had got the thermodynamics beautifully right once and for all, they would have let the matter rest there and saved us from subsequent problems. Instead, however, they abandoned thermodynamics which had served them so well, and invented bioenergetics. They did this by assuming that the free energy of hydrolysis of ATP had been transferred to the enzyme which, then, could transport Ca2+ against a concentration gradient. This has been generally accepted, but comes with two adverse consequences. First, it seems to say that this phenomenon (energy transduction) is exclusive to molecules of life, because it does not occur in normal thermodynamic systems and can only operate when thermodynamics has been abandoned. i.e. we have been saddled with a vital force, something which scientists have always been at pains to avoid. Secondly, it has discouraged attempts to elucidate the mechanism by which the enzyme and ATP

61

between them contrive to transport Ca2+ against a gradient. Who needs a mechanism when a transfer of energy explains everything? The free energy of hydrolysis of ATP, of course, consists of evolution of heat and increase in entropy. There is nothing there that can be reused in any way. Going back to the state of play before the birth of bioenergetics, transport of Ca2+ against a concentration gradient does not need a gift of energy: it can legitimately proceed as long as the concentration gradient is within the limits determined by the free energy of hydrolysis of ATP. Thus the calculations made to determine whether there is enough energy to support transport are correct, but the support consists, not of donation of energy, but of dissipation of energy as heat to compensate for the positive free energy change of the work performance. It is ironic that the much vaunted “energy currency of the cell” produces only hot air. These arguments apply to many, probably all, of the coupled reactions of enzymes. The work process may be synthesis of a molecule unstable in the mixture of LDW and HDW existing in bulk water, sliding of the filaments in muscle, transport of kosmotropic cations other than Ca2+. The common mechanism is induction of a pocket of LDW in which the reaction takes place spontaneously, followed by conversion of LDW into HDW with release of product. The coupling reaction is not always hydrolysis of ATP. All that is need is a spontaneous reaction with a large enough free energy change and a mechanistic involvement in the process of the work performance, usually a conversion of LDW to HDW and return of the enzyme to its ground state so that a new cycle can take place. When it comes to speculation about the origin of life we are back in the world of thermodynamics. Synthesis of peptides, polynucleotides and polysaccharides from precursor monomers does not need an injection of energy (as has been commonly supposed). All that is needed is a change of solvent and coupled reactions. And, of course, two-state water supplies both. In the example already given of synthesis of ATP in cellulose acetate membranes there was no injection of energy but there was present a pocket of LDW in which the reaction took place spontaneously and small chaotropes which diffused spontaneously into the LDW and converted it to HDW, releasing ATP. Prebiotic clays must have contained stable pockets of LDW in which synthesis of chiral polymers could take place spontaneously, and both chaotropic ions which would release products from proximity to hydrophobic surfaces and kosmotropic ions which would release products from LDW associated with fixed charges. Athough the experiment has not been done, it is possible that the first self-replicating polymer could be created at a suitable surface in the presence of an excess of monomeric units.

62

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