The surface energy of water: functional implications of hexagonal/cuboidal transformations in the surface arrays.

Hyde's scientific book The Language of Shape has emphasized the importance of minimum surfaces in the structure of biological membranes. Minimum surfaces can be visualized as the property which brings many droplets of liquids to spherical bubbles, since a sphere has the minimum surface to volume ratio. Thus, a sphere with a surface of 4pir2 and volume of 4/3pir3 has a surface to volume ratio of 3/r, that is, the ratio is dependent upon the reciprocal of the radius. The chemistry of water as dihydrides of the electronegative element oxygen is fundamentally dependent upon its polar properties and particularly the delta positive charges on the hydrogen atoms and the double delta negative charge on the larger oxygen atom, which from its mass (16 Da) is regarded as the centre of the water molecules. The cohesion of water as a liquid or as semi-crystal like structures in the surface depends upon electrostatic forces that are comparable in strength to covalent bonds. This review discusses the functional implications of some unexpected properties which have been evinced by model building and illustrated as a Poster in the 4th World Congress of Cellular and Molecular Biology.

The cohesion of water in biology: a property not to be forgotten.

Gases and crystalline solids are states of matter that have been understood for nearly two centuries but liquid as a state of matter is still unclear. As a third state of matter there have been many anomalies uncovered in the twentieth century such as dipoles and different properties from changed molecular structures. In consequence liquids can no longer be grouped into a separate state of matter. Liquid water, the most abundant material in the Universe has a number of discrete characteristics. The liquid surface and the cohesive forces of liquid water are two of the more general properties that have unique importance in molecular biology. It is shown here that Coulomb forces over short distances can stabilize water molecules that are rapidly spinning dipoles or dipoles which have lost their rotational energy and form semi-crystal-like solids when confined to the restricted spaces of cells and proteins. The surface energy of liquids, extensively studied since the 19th century, can do mechanical work and this is clearest with liquid mercury. But it is surprising that this remarkable property has been neglected in the case of water in biology, and particularly not envisaged as a work supplementary to ATP hydrolysis in muscle contractions, which merits further study.

The surface energy of water; molecular biology of the working mechanisms.

The surface tension and surface energy of water, have been studied for two centuries. The ability to do physical chemical work dates from the decades at the close of the nineteenth century and opening of the twentieth. Teaching and research popularity dropped in the post-war years, and today is practically unique. Before 1846, Laplace, the French scientist, found that pressure was needed to force water, or other liquid, through small holes. The pressure needed was greater for smaller holes, and the relationship depended upon the surface tension of the liquid under test, and the inverse of the radius of the holes. The Laplace formula still appears in Physiological textbooks. The importance in the small air tubes of respiratory physiology, and in the capillary circulation generally, is still studied. The use of surface energy for protein conformational changes has received little attention. A proposed scheme for the human erythrocyte glucose transporter, although shown to be experimentally consistent, has been disregarded. A possible reason is the difficulty of comprehending how surface energy can actually perform mechanical work. Therefore, studies with molecular models, which increase the understanding of how mechanical work is done are described in more detail here.

Biological energy sources: the surface energy and the physical chemistry of water. Examples from studies on muscle contraction.

The physical chemistry of water at nanometre dimensions was used to explain the conformational changes and water breaking properties of the glucose transporter protein (GLUTI) in human erythrocytes more than ten years ago. The energy for this hidden work arises from cycles of evaporation and condensation of water within the cells but was several times larger than resting metabolism. Physical chemical principles can quantify the hidden work done and demonstrate that a significant source of energy is available, which is free of the metabolic energy derived from the hydrolysis of ATP. Therefore, a more widespread biological use of this "free" energy source was probable and a working hypothesis, which applied this energy to supplement the work derived from ATP hydrolysis in muscle, was proposed. The scheme gives a complete explanation for the unexpected and novel findings in skeletal muscle reported from Italy. The problem of using two energy sources and the novel properties of water at nanometer dimensions as they would apply in muscle are briefly discussed but they merit further interdisciplinary studies.

The surface energy of water: the largest but forgotten source of energy in biological systems.

Many functional proteins perform mechanical, structural or chemical work. Such proteins often use the energy from the hydrolysis of adenosine triphosphate (ATP). The role of ATP as an energy source and its production by metabolism was established in the middle of the twentieth century and replaced glycolysis as the focus of study. Before this time the surface energy of water, quantified in the middle of the nineteenth century, had been visualized as an important source of biological energy. Experimental and theoretical work has shown that the internal work done by this energy source may greatly exceed the energy derived from metabolism. Although the energy from ATP usually does the work external to the body, even this may be supplemented by the surface energy of water to give greater efficiency. The consideration of the principles by which proteins might employ this larger source of energy to do work is germane at this time.

The sequestration of hydroxyl ions by C2 in liquid water: useful physiological roles for a reversible complex formation in the presence of protein catalysts.

A second function of carbonic anhydrase (CA) isoforms has already been proposed. This involves the dispersal of complexes in which six carbon dioxide molecules sequester a hydroxyl ion when the gas reacts with liquid water. The semi-catalytic reaction does not require the formation of bicarbonate as an essential corollary. This function is, therefore, a likely activity of carbonic anhydrase related proteins that have recently been discovered and which lack the active zinc site essential for the hydration of carbon dioxide. Re-examination of possible functions for the complex of six CO2 molecules with a hydroxyl anion have brought to light several circumstances where the presence of fully reversible complexes could have physiological advantages. A catalytic synthesis and dissolution of the complexes could thus be the important function for the carbonic anhydrase-related proteins (CA-RP) molecules as well as of some CA isoforms. The possible mechanisms for this extended second catalytic function and examples are briefly discussed.

The sequestration of hydroxyl ions by CO2 in liquid water: the physiological implications and the second function of carbonic anhydrase.

The pH changes due to bubbling CO2 through water produced anomalies which were more readily explained by an hypothesis based on electrostatic attractions between the molecules. The present studies have suggested that an hexagonal array of six carbon dioxide molecules could bind and sequester a hydroxyl anion. The binding energy of the complex is estimated to be comparable with that of a covalent compound and its dissociation may only occur at the water interface with air or at the water/hydrophobic protein interface in a protein cleft. The physiological importance lies in the consequential release of an equal number of free hydrogen ions (H3O+) and the disruption of the normal action of buffer systems in regulating the cytoplasmic pH. The counteraction of this sequestration reaction and the acid-base disturbances which result, form the second important function of carbonic anhydrase isoforms, the mechanisms of which are briefly discussed.

The physiological properties of human red cells as derived from kinetic osmotic volume changes.

Volume changes were originally used for studying the dynamic properties of glucose transport in red cells. As an extension it has been found possible to examine the interplay of three functional proteins evolved for the physiological role of human erythrocytes in transporting carbon dioxide and bicarbonate. The proteins chiefly concerned in this investigation were the cytoplasmic carbonic anhydrase and the two membrane transporting proteins, namely the band 3 anion exchanger and the unique bicarbonate transporter, which are distinct from the anion exchanger. The rates of anion membrane transport measured and the volume changes may be more than two orders of magnitude faster than those which regulate cationic movement in red cells, but this may only be an adaptation for the physiological role of red cells. The new concepts derived from the studies and their possible wider applications to physiological mechanisms are briefly discussed.

The triphasic volume response of human red cells in low ionic strength media: demonstration of a special bicarbonate transport.

When human red cells are first placed in isotonic media of low ionic strength there is a triphasic volume change. The initial shrinkage (phase A) starting immediately is followed by a rapid reflation (phase B). The final shrinkage (phase C) is slower and has more variable properties. The first two phases need Cl- and HCO3- and they have been shown to involve both the band 3 anion exchanger and the red cell carbonic anhydrase. Phase C is only seen when phase B is present. Both phase B and phase C were accelerated by the lower monohydric alcohols. With methanol the acceleration was maximal near 2M, thereafter inhibition developed. The hypothesis is advanced that phase C involves a permeability for bicarbonate which is independent of the band 3 anion exchanger, and which may be a dimer of carbonic anhydrase. The unique kinetics, the physiological significance, and implications of the special bicarbonate transport system are discussed.

The acceleration of pH volume changes in human red cells by bicarbonate and the role of carbonic anhydrase.

The red cell shrinkage rate due to bicarbonate in media of high pH (ca 9.4) has been compared with the hydroxyl shrinkage rate on a per mM basis. The shrinkage rate due to bicarbonate was only half that due to OH-/Cl- exchanges. It was therefore deduced that the Jacobs-Stewart cycle was limited by the carbonic anhydrase step and not by the rate of transport on the anion exchanger protein. To explain this and other anomalies the hypothesis is made that carbonic anhydrase has evolved as a pH-dependent catalyst with specific physiological functions in pH regulation and in other cellular mechanisms. The kinetic theory and some physiological implications of the hypothesis are discussed.

The physiological properties of the band 3 anion exchanger as derived from pH and other studies with human red cells.

The kinetics of the erythrocyte anion exchanger which have been studied at high pH values have confirmed the importance of hydroxyl ions in the overall physiological roles of the band 3 protein in human red cells. Although usually characterised as a one-for-one anion exchanger, conditions have been identified which give the appearance of net transport. The present findings have uncovered new functional features consistent with the properties of anion transport in other tissues. The molecular models proposed to interpret the results could have wider applications to other cellular mechanisms. Some of these are discussed to illustrate the physiological implications which can be deduced from the novel kinetics of this unique membrane transport protein.

An hypothesis for a contractile protein based on the principles of submicroscopic physiology.

The discovery of the polyguanidinium ring complex and its various properties has emphasised the versatility of arginines in functional proteins. In an extension of these studies the possibility of formulating a polypeptide which could be the basis for a contractile element in muscle presented itself. Although hypothetical, the deduced molecular properties, based on the unique chemistry of arginine guanido groups are in keeping with known functional aspects of some muscles, and could form the foundation for a more detailed theory of muscular contraction. The structural requirements and the theoretical principles involved are briefly described in this paper.

The pH volume changes of human red cells in vitro due to the exchange of chloride and hydroxyl anions.

In glucose exit experiments measured photoelectrically, the excursions on the chart recorder were found to be larger for exits in media of alkaline pH. This was shown to be due to the addition of a pH volume effect to that of the osmotic shrinkage resulting from the glucose efflux. The pH-dependent volume change also occurred in glucose-free cells and was a linear function of the pH of the medium between pH 6.8 and 9.0. The effect is consistent with the loss (or gain) of chloride in exchange for hydroxyl anions on the band 3 anion transporter and with the buffering of the hydroxyl anions by haemoglobin. The implications for the working of the anion exchanger and for respiratory physiology are discussed.

The polyguanidinium complex of the glucose transporter: a possible basis for bistable cationic gates and anionic channels in biology.

The detailed molecular structure and mechanism of action of the red cell glucose transporter would endow it with the ability to transfer the less hydrated potassium ion. This is prevented by a polyguanidinium-ring-complex cation which provides an effective electrical energy barrier. The ring complex has structural features which could form a bistable state of ring charges; in one form the electrical repulsions would be fully effective (gate 'shut'), whereas in the other form they would be ineffective (gate 'open'). It is also suggested that the polyguanidinium linkages could form a linear complex which could be the basis for anionic channels in biology. The theoretical basis for both these hypotheses is described.

The role of the surface energy of water in the conformational changes of the human erythrocyte glucose transporter.

There is now strong experimental evidence that the red cell glucose transporter protein operates by a conformational change which has the effect of presenting a sugar binding site to the outside and inside medium of the cells in an alternating manner, but the way in which this is brought about is still unknown. Kinetic evidence that the conformational changes which create the inward and outward facing modes can occur in the absence of a sugar substrate has been put on a firmer experimental basis by Appleman and Lienhard (1989). Conformational changes of the magnitude envisaged are too large to be attributed only to spontaneous thermal agitation in the protein. Theoretical considerations show that the surface energy of water could be an alternative source of energy which, in a suitable reciprocating cycle of activity, could sustain the conformational changes throughout the existence of the transporter.

The polyguanidinium-ring-complex cation shield in the human red cell glucose transporter.

Widdas and Baker (1991) have described a possible structure and functioning of the human erythrocyte glucose transporter, based on its amino acid sequence. It was noted that eight positively charged side chains surrounded the proposed transfer cleft on the inside. These could form a ring shield which prevented the loss of K+ ions. Theoretical and model-making considerations point to this shield being made up of a polyguanidinium-ring-complex cation with eight (or ten) positive charges distributed over two identical rings of hydrogen bonds and normal linkages.

The accumulation of free and phosphorylated sugars in adipocytes based on a dynamic diffusion barrier.

The simple theory of a dynamic diffusion barrier is described and it is shown how this could account for the accumulation, in adipocytes, of those free sugars which are also phosphorylated. The standing concentration gradient established by this mechanism depends on the recycling of free sugar and sugar phosphate in submembrane structures which start in juxtaposition to conventional membrane hexose transporters. Although a continual expenditure of metabolic energy is involved, there can be a net gain from the potential-energy store of accumulated substrates. The hypothesis leads to a series of simple equations which can be used as the basis for computer simulations of experimental procedures.