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Feature

Weird, Wonderful Water

Everyone knows water is plain old H2O, but nothing's that simple...

by Professor Philippa Wiggins

Water is unique among liquids. Its unique chemical and physical properties have played a vital role in the development and maintenance of life on this planet.

The deceptively simple chemical structure of water, consisting of two hydrogen atoms joined with strong covalent bonds to a single oxygen atom, holds a clue to its unusual features. The whole molecule is bent into a tetrahedron, with the oxygen at the centre and the hydrogen at two corners.

Directed toward the other two corners of the tetrahedron are the oxygen's "spare" electron pairs, producing a molecule with two positive poles where the hydrogen atoms are and two negatively charged poles where the electrons are found.

This distribution of charges means that each water molecule attracts others. The hydrogen atoms are weakly attracted to the oxygen atoms of neighbouring molecules to form a network of hydrogen bonds. Unlike all other liquids, water exists as three-dimensional networks of mutually hydrogen-bonded molecules.

Although bonds in these networks break and reform 1012 times per second, a "snapshot" would reveal a continuous pathway of bonding throughout the entire volume of the liquid. A glass of water is, effectively, one giant molecule.

Although the individual hydrogen bonds are rather weak, they add up to a force which dominates the physical and chemical properties of water, setting it apart from all the other covalently bonded oxides and hydrides of the first row of the Periodic Table. All the others --  such as methane (CH4), carbon monoxide (CO), and nitrogen dioxide (NO2) -- are gases or decompose at room temperature.

When the hydrides of Group 6 of the Periodic Table (to which oxygen belongs) are compared, water stands out as an oddity. Plot the melting and boiling points against the molecular weights, and the hydrides of sulphur, selenium and tellurium behave in an intuitively acceptable manner: as the molecules get heavier, more energy is needed to free them from the solid into the liquid and from the liquid into the gas, and thus melting points and boiling points increase.

Extrapolate from these, and the hydride of oxygen -- H2O --  should melt at -95oC and boil at -75oC. But, of course, water melts at 0oC and boils at 100oC. As illustrated in the graph below, the hydrogen-bonded network has raised its melting point by 95oC and its boiling point by 175oC.

Clearly this is no trivial influence. Change the strength of this network and all the properties of water change with it. Water can be "stretched" by moving the molecules apart from one another to produce a low density form; it can also be "compacted", by pushing the molecules together. These simple changes in density have a surprisingly profound effect upon the network.

Stretched and Compacted Water

In normal liquid water, molecules are too close together to make many straight, strong hydrogen bonds in which a hydrogen atom lies on a straight line between two oxygen atoms. The bonds that do form range from straight to very bent, or from strong to weak. In stretched water, the number of straight bonds is greatly increased; compacted water has relatively few straight bonds.

Such changes in the density of water are extremely common in association with solutions or gels of biopolymers, large biological molecules such as proteins.

As a solvent, water enables substances to dissolve and spread throughout the solution; such dissolvable substances are called solutes. When added to water, many solutes break apart into their component ions. Thus salt, or sodium chloride (NaCl) dissociates into positive ions, or cations, from its sodium component (Na+); and negative ions, or anions, from its chloride portion (Cl-).

These processes, and changes in water density, occur at their simplest where charged regions of a protein surface, or a cell membrane, come into contact with a water solution. Each positive or negative charge on the protein is balanced by a counter-ion in the nearby solution. There are two different populations of water present: one near the charged surface, which has a high concentration of counter-ions; and a second zone, away from the surface, which has no dissolved ions present. The water molecules of these two populations freely exchange with each other.

In this case, the water can't simply move into the area of strong ionic concentration to dilute it and produce a solution in equilibrium throughout. The electrostatic attraction of the protein's charged groups is too great for the counter-ions to spread out evenly in the solution.

Usually, water will move from a more dilute solution to a more concentrated one until equilibrium is reached. When this movement happens through a semipermeable membrane, such as a cell membrane, it is called osmosis. Classical osmotic theory says that the pressure of water on the zone of water containing the counter-ions increases the chemical potential of that water, so that the two populations of water molecules equilibrate. Presumably there is a pressure, but experimental evidence has shown such solutions and gels also contain stretched water.

Apparently pressure is not enough, and in order to equilibrate, the two populations of water molecules must also change their densities. Molecules of water away from the regions of high ionic concentration move apart, decreasing their local chemical potential by doing work of expansion to produce stretched water. The molecules near the charged protein surface compact, increasing their local chemical potential. Thus is equilibrium established.

A second place to find stretched water is adjacent to a hydrophobic or weakly hydrogen-bonding surface. Water at such a surface is in a state of higher energy than more distant water molecules which can make relatively strong hydrogen bonds with one another. In order to equilibrate, it stretches to lower its chemical potential to that of the bulk water nearby.

The building blocks of cells -- proteins, polypeptides, polysaccharides and polynucleotides -- all contain both charged and weakly hydrogen-bonding patches of surface. Much of the water associated with them in solutions or gels is either stretched or compacted.

If stretched water is as common in gels and polymer solutions as has been suggested, why has it not previously been recognised?

Elusive Target

There are several reasons why it has been overlooked. In a polyelectrolyte solution or gel, stretched and compacted water co-exist. Any water-related property measured globally on the whole solution or gel, therefore, gives an average value which is not necessarily very different from the value for normal water. Often at a hydrophobic surface, there is relatively little stretched water, and its properties are swamped by those of the more abundant normal water.

When excess electrolyte, such as salt, is added to a solution in contact with a charged protein surface, the salt moves preferentially into the compacted water zone. This results in an increase in the amount of compacted water present. When a solute which prefers stretched water is added, the stretched water equilibrates by compacting and reverting to its normal density, structure and solvent properties. Both responses make it difficult to detect the stretched water present.

A third reason for the failure to detect stretched water has been the general belief that the effect of osmotic pressure is enough to explain equilibration in biopolymers. There has, therefore, been no motivation to look for stretched and compacted water in association with biopolymers. A subtle phenomenon is unlikely to be discovered until there is a theoretical expectation that it might exist.

Biological Significance

It is the secondary effects of the changes in density associated with stretched and compacted water that are biologically significant.

As the water stretches and the network expands, hydrogen bonding becomes stronger, the liquid more viscous and diffusion through it slower. The water becomes less reactive, as more free OH groups and lone pairs of electrons become engaged in stronger hydrogen bonds. Of greatest import as far as biochemical mechanisms are concerned, the behaviour of water as a solvent changes in a highly selective manner.

The selectivity depends on the nature of any ions in the water solution. Cations are positively charged ions; anions are negatively charged. Small, highly charged cations, such as Mg2+ and Ca2+, will move preferentially from stretched water into normal water, and from normal water into compact water. Larger cations, such as NH4+, will seek out stretched water where possible. Predominantly hydrophobic solutes, such as propanol or ethanol, go selectively into more weakly hydrogen-bonded, compacted water. Some amino acids, glucose, urea and betaine prefer stretched water.

The cyclical making and breaking of stretched water is the very essence of the cyclic behaviour of important cell mechanisms such as enzymes, which act as catalysts for many reactions, and ion channel entrances, which govern the movement of ions across cellular membranes. Ion channels are particularly important, for example, in the electrochemical functioning of nerve cells.

Interactions between these proteins and their specific electrochemically important counterparts have evolved over millions of years to put the timing of the switches in water structure under tight control of the functional protein itself. Moreover, in all such processes the making and breaking of stretched water has a directional component, which explains many enzyme mechanisms.

Pumping Ions

Transporting ions across a membrane requires energy, particularly when this occurs against a concentration gradient. For this reason, such mechanisms involve the energy-carrying molecule adenosine triphosphate (ATP). ATP is split, or hydrolysed, by special enzymes known as ATPases to release energy. When this happens, a phosphate group is released and is taken up by another compound in a process known as phosphorylation.

Cation ATPases can pump cations across membranes against concentration gradients, using the special properties of stretched and compacted water. The active centre of the pump contains negative sites, specific to the positively charged cations being transported, and an aspartyl group, also negatively charged.

All these charges are first neutralised by counter-cations. The cavity, therefore, contains weakly-bonded, fluid, compacted water into which small cations readily diffuse and bind to their specific sites. The counter-cations, no longer needed for electroneutrality, diffuse out, taking some water with them. The compacted water relaxes its degree of compaction, becoming more normal.

A form of ATP, called MgATP, enters the now nearly neutral cavity and phosphorylates the enzyme, removing the last negative charge. The cavity is now highly hydrophobic. Water starts stretching at its interface with the external solution and spreads upward toward the apex of the cavity, driving small, highly charged cations ahead of it. When the cations reach the channel opening at the apex, they diffuse out into normal water on the other side of the membrane. In order to maintain electroneutrality, counter-cations re-enter from the cellular solution and water reverts to its compacted state. The phosphorylated enzyme hydrolyses in this more reactive water, restoring the last negative charge.

The several steps of this sequence of reactions are all inevitable consequences of the preceding steps. ATP cannot enter the cavity and phosphorylate the enzyme until binding of cations has reduced the high negative charge. Water does not regain its reactivity until the transported cations have moved out through the channel -- cations both prevent the channel opening too soon, and close it behind themselves. This mechanism is valid for any small, highly hydrated cation. Specificity lies only in the binding sites where the reaction takes place.

Cation ATPases are examples of a unidirectional spread of stretched water pushing highly hydrated solutes through a channel. Some hormones, on the other hand, have ionic groups which are attracted to the stretched water and so are drawn into receptor cavities. Since, however, these ionic groups carry counter-cations with them, even when the hormone binds to a specific site, the activity of water inside the cavity decreases relative to that outside.

If the cavity is rather rigid, so that water cannot diffuse in to equalise, the stretched water must eventually collapse back to its normal density and solvent properties. The oxyanionic groups are now in an unfavourable environment; the hormone dissociates and leaves the cavity. The time between binding of the hormone and its dissociation must obviously be long enough to produce the desired biological response, but not much longer. Again, timing is crucial and is built into the overall mechanism.

These two examples illustrate how stretched and compacted water and their mutual interconversions are probably exploited biochemically. Macromolecular evolution took place in this unique solvent and, from its beginnings in watery clays. had to contend with spontaneous solute-induced changes in the strength of water's hydrogen-bonded network.

Evolution has apparently favoured proteins which can control the switches in water structure. These switches have been built into the control mechanisms of the basic catalytic processes which drive all cells. Thus the unique properties of stretched and compacted water --  and its utilisation in this fashion -- can be considered one of the driving forces of life.

Professor Philippa Wiggins is in Auckland University's School of Medicine.