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Volume 82, 1954-55
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Public Lecture
The Struggle Against Equilibrium—A Physico-Chemical Problem in Life

Division of Food Preservation and Transport, C.S.I.R.O, Sydney, Australia.

Few problems in science have excited so much speculation as the nature and origin of life. Not only have biologists, who are legitimately concerned with investigations of the nature of living matter, thought and wondered about this problem, but also physical scientists, physicists and chemists have added their interpretations. Thus in recent years, we have seen physicists such as Schrodinger writing “What is Life?” and Bernal “The Physical Basis of Life.” The distinguished physical chemists, Linus Pauling and Sir Cyril Hinshelwood, have devoted much of their recent work to biological problems. Organic chemists such as Sir Robert Robinson and A. E. Todd have worked on molecules of biological origin. Despite all these investigations of biological problems we are even more aware of the complexity of the living system and of the difficulty of defining “life” itself. Ever since N. W. Pirie wrote his well known essay on the “Meanniglessness of the terms of living and non living” there has been a noticeable coyness about defining the term “life.”

I have chosen to talk to you this evening on one attribute of living matter the ability to prevent the system from coming to equilibrium, for, however difficult it may be to define life, this is certainly one of its most characteristic attributes I think every scientist would enjoy reading Schrodinger's “What is Life” Here you will find this characteristic of living matter expressed in words which will probably appeal to you. If you are a chemist you may be pleased with the statement that living matter “evades, the decay to equilibrium”; if you are a physicist you may appreciate the view that living matter “feeds on negative entropy, i.e., unlike non-living matter extracts order from its environment”; and if yon are a biologist you will be relieved to hear that this means no more than you have known all along— that living matter receives energy, uses energy, does work and dissipates the energy in so doing. Living matter is never in equilibrium; it is always living on the flow of energy—for life is like a water wheel turning as long as the stream of energy passes it and ceasing when the stream ceases.

But I do not want to talk about abstract views or speculations on the nature of life. When your Committee asked me if I would give a technical lecture to the members of the Congress, I decided to take a particular example of the way in which living matter is constantly struggling against the dead hand of the equilibrium and to interest you in a physico-chemical problem which affects all of us and interests many of us. The other reasons for the choice were that we have been active in research contributing to this field and that it offers an example of the quantitative approach to biology.

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The problem to which I refer is the physiological process whereby living cells maintain their content of salts and other ionised compounds or actually secrete high concentrations of ions. The problem then is how living cells pass electrolytes into themselves and hold them in high concentration (a process we term salt accumulation) or pass electrolytes through themselves (a process we term secretion). Why all this interest in the movement of salts into and out of cells? First, because every living cell always has a problem of holding these substances or of passing them across. Second, while we do not know how every living cell is affected, we do know that this process of active transport (secretion and accumulation) is responsible for a variety of the processes on which our lives depend, e.g., secretion in pancreas, stomach and kidney, organization of nerves, blood corpuscles and so on. Further the regulation of blood can position in fish and other aquatic animals, and the accumulation of electrolyte in plants, the basis of mineral nutrition, depend on these properties of living cells.

Although the cell theory—the hypothesis that all living organisms are composed of cells—is only a little over a hundred years old, this has been so substantiated as to be no longer a theory and is indeed common knowledge, not specialised knowledge of biologists. While you will all realise that living organisms are composed of a wide variety of cells you have probably not thought very much, unless you are a biologist, about what size the cell is, and about what its problems of maintaining substances in solution may be. Cells vary greatly in size, shape and function but if we talk of a typical plant cell for a moment we are thinking of a body which is about 60 microns in diameter, i.e., about 1/16th of a mm. and which is merely a small gelatinous sphere of material surrounded by a cellulose wall, which having the consistency of blotting paper is freely permeable to water and all substances which dissolve in water. Inside this wall is the gelatinous mass of living material, the protoplasm, surrounding a central sphere called the vacuole which contains non-living material and only substances in true solution. Inside this tiny sphere of living matter which you can hardly see with your naked eye, the cell controls the entry of the dissolved substances which it requires, prevents the loss of others and excretes substances which it does not require any longer. Seeing that the composition of the cell may be extraordinarily different from its surroundings this requires a considerable sub-microscopic organization. Frequently in elementary studies of cells this capacity to “hold their own” is dismissed by reference to a membrane which is a barrier to the diffusion of the substances from the cell. But a moment's thought shows that it must be a good deal more complicated than that. The cell cannot have a barrier impermeable to substances which it needs to retain and, at the same time, permeable to the substances which must pass in from the exterior. Further, no membrane even if it does exercise some resistance to the movement of substances in solution will be perfectly impermeable. When you consider that the membranes which these minute cells can organize are probably only 100 to 300A in thickness, yon will realise that their impermeability is purely a relative term. Within an organization of this type therefore we must look at the cells capacity to resist the tendency for the interior of the cell to come to equilibrium with the external medium.

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Instead of giving you a number of examples of the differences between the interior and exterior of typical living cells, I want to discuss in detail just two examples and to suggest how the rather spectacular differences between inside the cell and outside, or between one side of the cell and another may be maintained. It seems a far cry from the stomachs of animals to the roots of plants but in the words of the Scriptures “all flesh is as grass” and physiologists find a surprising similarity of all organisms. For this reason I want to talk particularly about the secretion of hydrochloric acid by the gastric mucosa, the cells which line the stomach, and the accumulation of salt in carrot tissue. Let me define these problems more closely; the lining of the stomach or gastric mucosa consists of a number of cells in a somewhat complex organization. Certain of these cells which are flask shaped and about 50 microns (1/16 mm.) in diameter are responsible for the secretion of hydrochloric acid on one side (the stomach side). The concentration of this HCl is very high (0·13M) so the pH is very low, about 1. This high concentration of HC1—more than 1/10 Normal—is presumably responsible for the advertisements for indigestion remedies, which have appeared in Australian papers, pointing out that the stomach creates an acid which will burn holes in the carpet! This high concentration of acid as hydrogen ion is about 3 million times greater than that in the blood which is on the other side of these small cells. I think you will agree that a physical chemist faced with the task of suggesting a mechanism for secreting hydrochloric acid from the low concentration of hydrogen ions at pH 7 in the blood to the high concentration of hydrogen ions at pH 1 in a space of 50 microns, would have a difficult task. How this concentration of acid is achieved is one of our problems for discussion.

The problem of accumulation in plant cells is different. I have already spoken of the structure of a typical plant cell. Since it is difficult to work plant cells of this size isolated from the tissue we usually work with discs or slices of tissue or segments of roots. When such a piece of tissue is placed in a dilute solution of a single salt such as sodium or potassium chloride, salt enters rapidly in the initial period and adjustment of ion concentrations, ion exchange, between the insides of the cells and external solution takes place. This in itself is a fairly complicated process which leads to the establishment of an equilibrium between internal and external concentrations, known as the Donnan equilibrium. Many of you will be familiar with different types of Donnan equilibria, some occurring in non-living media such as colloidal suspensions of clay or other particles and ion exchange resins, but I do not wish to discuss this in detail to-night. After this initial physical adjustment between internal and external concentration, salt continues to enter the cell until the concentration inside is greater than the outside concentration and still salt enters at a steady rate. Eventually this process which may continue for days builds up a very high internal concentration compared with the external. For example, 14g of carrot tissue in the appropriate conditions will absorb practically all the salt from 240 mls of 0 005 M. KCl Thus the internal concentration rises to about 17 times the original external, by which time the external concentration is very low (only about 10-4M) so the final internal concentration is about 1,000 times the final external. Incidentally slices of carrot tissue will absorb sufficient ions from a dilute solution to reduce the electrical conductivity of that solution to about 2 × 10-6 mhos/cm. which is

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of the same order as good distilled water—a unique method of obtaining pure water by a biological process; I have not yet patented this method! Clearly one characteristic of this process is that both anions and cations of the salt supplied are absorbed by the tissue.

The rate of accumulation, i.e., the amount accumulated per unit time after the initial uptake is proportional to the concentration in very dilute solutions but becomes independent of concentration as the concentration increases; this means that the ratio of internal to external concentration is not important in governing the rate of accumulation. The rate does fall eventually with time suggesting that, as might be expected, the more salt that has been accumulated the more difficult it is to accumulate more. Ions from different salts are accumulated at different rates. K+, Na+, NH4+, Rb+, Cs+ are accumulated rapidly. Li+ less rapidly and Ca++, Mg++, Ba++ only slowly. The anions NO3-, Br- and Cl- are accumulated rapidly, while the divalent SO4- is hardly accumulated at all.

I have now defined two living processes; one in which hydrochloric acid is secreted in high concentration and the other in which salt is accumulated against the concentration gradient. The living cells concerned are working against the attainment of the equilibrium which would be achieved by diffusion of these ions to equality of concentration on both sides. Some years ago I was lecturing on this subject in Sydney and had just reached this point, when Dr. Nieholm who is now Professor of Physical Chemistry in the NSW University of Technology, said to himself—“Heavens, here is a man who is going to break the second law of thermodynamics”—what he probably thought was—“Here's a poor ignorant botanist with even more ignorant plants which have never heard of the second law of thermodynamics.” Perhaps some of you are beginning to wonder about the same thing. Let me put your minds at rest at once by saying that biologists are not forgetful of this law and that I hope that we shall be able to show that even plants and animals do not break it. We must all agree with that great man of science, Sir Arthur Eddington, who said that the second law of thermodynamics holds the supreme position among the laws of nature Perhaps yon remember, he goes on “if someone points out to you that your pet theory of the universe is in disagreement with Maxwell's equations—then so much the worse for Maxwell's equations If it is found to be contradicted by observation—well, these experimentalists do bungle things sometimes. But if your theory is found to be against the second law of thermodynamics I can give you no hope; there is nothing for it but to collapse in deepest humiliation.” We biologists cannot offer you anything as picturesque as the demon which Clerk Maxwell suggested would overcome the second law of thermodynamics. There is no little man in living cells slamming frictionless doors behind fast moving ions; there is no contravention of the second law of thermodynamics. The cell achieves this is disequilibrium by the expenditure of energy and our problem, therefore, becomes how the cell provides energy in a form to do the work necessary to accumulate these ions or secret the acid.

The process in living cells which supplies energy to other activities where work must be done, is of course, the process of respiration and when this process of accumulation and secretion had been clearly recognised it was natural to look for the connection with respiration. In this, carrot tissue, which has been the guinea pig of our laboratory, for a number of years, is particularly

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useful. When slices of carrot tissue which have been respiring for some time in water are transferred to salt solution, the oxygen uptake increases, sometimes by as much as 100%, and the rate of salt accumulation is directly proportional to the increase in respiration, whether this increase is varied by variation in temperature, in concentration of salt or in the nature of the cation in the solution. This increased respiration is termed salt respiration and, as has been shown, can be completely inhibited by carbon monoxide in the dark but this inhibition is reversed by light. Now this property of inhibition by carbon monoxide is a specific character of one particular enzyme of the respiration process, an enzyme known as cytochrome oxidase and in order to understand the significance of this we must discuss very briefly the nature of the process of respiration. In normal respiration of plants and animals the sugar is oxidised according to the following equation: C6H12O6 + 6O2 + 6H2O = 6CO2 + 12H2O + energy. This of course, takes place in a large number of steps (about 30 of them) which we need not discuss now. I do want you to note however, that the hydrogens liberated from the sugar and from water eventually combine with oxygen to form water. Before they do so they are separated into electrons which reduce the substances in the cytochrome system, and hydrogen ions which presumably are dissociated or are capable of dissociation from the cytochrome. This reduction of the cytochrome takes place by a change in valency on the iron atom which the cytochrome molecule contains, and eventually the electron from cytochrome oxidase together with a hydrogen ion is passed over to the oxygen with the formation of water. From the equation every time 6 oxygen molecules are taken up in respiration, 24 hydrogen ions and 24 electrons must be handled by the cytochrome system. In other words, one oxygen molecule requires the cytochrome system to pass 4 hydrogen ions and 4 electrons to form water. The significance of this will appear in a moment.

The observation that inhibitors which cut out the salt respiration also prevent accumulation was first made by Professor H. Lundegårdh, the distinguished Swedish plant physiologist. It occurred to Lundegårdh that the cytochrome which is capable of behaving as an electron carrier might also behave as a carrier of an anion and that the process might be the carriage of anion

Picture icon

-Schematic representation of the electron and anion transport system.
Solid lines represent chemical reactions; broken lines represent movements of substances. (Fe+++, oxidized cytochrome; (Fe++ reduced cytochrome.

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inwards each time an electron came outwards. Since the cytochrome is capable of a change of valency, if it brought an electron out to the cell surface in the ferrous form, it might have unit positive charge and take an anion, for example, chloride, into the cell attached to the ferric form. Meanwhile the hydrogen ions which had been liberated during the process of respiration at the cytochrome system might move out of the cell on other paths for positive ion carriers and the cation from the external solution, for example, potassium may move in on the carriers which had brought the hydrogen ions outwards. This whole process can be best understood by reference to the diagram. Lundegårdh's suggestion therefore means that the carrier for anions inwards, is the carrier for electrons outwards, and the carrier for rations inwards, is the carrier for hydrogen outwards. The cytochrome system possibly offers a reasonable hypothetical carrier for the anions.

In my laboratory, we had arrived independently at a hypothesis similar to that of Liundegårdh and we saw that one test of the hypothesis would be whether the amount accumulated bore the right quantitative relation to the number of electrons and hydrogen ions traversing the cytochrome system. If Lundegårdh were right, clearly the maximum rate of accumulation of a monovalent anion could not exceed the number of electrons traversing the cytochrome system at the same time. Now since each g. mol of oxygen taken up in respiration would require 4 g. mols of hydrogen ion plus electron, the maximum rate of accumulation of a salt would be 4 times the oxygen uptake in g. mols. In collaboration with Miss Wilkins, I was able to show that the maximum rates approach but do not exceed this hypothetical ratio of 4 times the oxygen uptake. Lundegårdh's hypothesis was therefore quantitatively feasible for plant cells.

Now what about the gastric mucosa? About this time the work on gastric acid secretions had reached the stage where the source of hydrogen ions was a subject of active investigation and valuable contributions had been made in recent years by Teorell, Conway and Davenport.* This work was accelerated very considerably by the development of the technique due to R. E. Davies of Sheffield, for measuring in frogs' stomachs, which continue to secret acid after isolation from the animal, the simultaneous oxygen uptake and acid secretion. The amount of acid secreted in the early experiments was about 4 times the oxygen uptake when both were expressed as g. mols. We can therefore picture the secretion of hydrochloric acid by the stomach cells as being the result of approximately half of the system required for the accumulation of salt, the difference being that cells secreting acid lose H+ ions to one side and do not swamp H+ ions for the sodium which is in the blood. Incidentally the passage of electrons to oxygen without their H+ ions results in the incomplete formation of water so hydroxyl ions are formed instead. This accounts for the observed fact that anions in the form of bicarbonate pass back into the blood in equal quantity to the hydrogen ions passing to the stomach. These bicarbonate ions are formed by the interaction of the hydroxyl ion with carbon dioxide and the enzyme carbonic anhydrase. I should mention here that Davies most unfortunately confused this explanation of the production of the

[Footnote] * See “The Biochemistry of Gastric Acid Secretion,” by E. J. Conway (Charles C. Thomas, Springfield, Illinois), 1953.

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hydrogen ion by some experiments in which he suggested that the hydrogen ion might be as high as 12 times the oxygen uptake instead of 4 times the oxygen uptake. This figure of 12 had always seemed rather uncertain to me because of the difficulty of estimating accurately the respiration actually engaged in the secretion and has been shown to be wrong by Davenport of the University of Utah, who explains how Davies' results were misinterpreted Davenport reaches the conclusion that the hydrogen ion production is certainly no greater than 4 times the number of oxygen molecules involved. Davies himself expressed some doubts about the interpretation of his results.

We have then the possibility from the quantitative data that the hydrogenions of hydrochloric acid secretion and the separation of the positive and negative charges which can be swapped for the cations and anions respectively in salt accumulation come from the respiration. The fact that these systems could be accounted for quantitatively on this basis suggests that there is here some fundamental principle which underlies the mechanism of salt movement in tissues as widely separated in evolution as the accumulating cells of plants and the stomachs of higher animals. Interesting as this hypothesis of the generation of positive and negative charges is, we must look further before we can arrive at a hypothesis to give a complete explanation of the two processes. For instance we need to know why the hydrogen ions diffuse with chloride to the lumen of the stomach and the bicarbonate ions diffuse back to the blood, balancing the sodiums which have been left by the chlorides. Again in plant cells, what keeps these positive and negative ions apart? How do the hydrogen ions swap for the cations and not form water until this is complete? We must examine the organization of the cells which makes these processes possible; here our knowledge is far from adequate. There are, however, some interesting facts of observation.

You will remember that we have decided that the enzyme system responsible for the generation of these hydrogen ions and their separation from the electrons, is the cytochrome system and the enzyme cytochrome oxidase is involved. Within the last 4 or 5 years we have obtained a very large amount of information on the location of the enzymes concerned with respiration within plant and animal cells It is now quite definite that the enzymes responsible for most of these steps in respiration are located in minute but definite particles in the cells. These particles, which have been known to cytologists for many years, are termed mitochondria and seem to have almost universal occurrence. Mitochondria, however, are about 1 micron in diameter and since the limit of visibility with the light microscope is about 0.3 microns, it is little wonder that the structure and functions of mitochondria have been little understood.

In recent years two techniques which have been developed rapidly, have made an enormous difference to our knowledge of mitochondria. The first of these is the biochemical technique of differential centrifugation which makes it possible to separate particles of a given size from broken cells, rather like cream is separated from milk in a separator. The particles of definite size, for example, nuclei and mitochondria, can be separated from each other by centrifuging for varying times at different speeds. Provided that two precautions are taken, the isolated mitochondria will preserve their respiratory activity so that they can be investigated after separation from the cells in which they were formed. The necessary precautions are that the temperature

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during the preparation must be kept low, i.e., should not rise above 5°C, and that the mitochondria should be kept in a solution of high osmotic pressure. If this is not done, the mitochondria swell, break and lose their activity.

The second technique is that of the electron microscope which has been invaluable in characterising small structures from cells. Quite recently the electron microscope technique has been extended very greatly by embedding material in plastic from which sections can be cut as thin as 200A; subsequently the plasic can be dissolved away and the sectioned material can be examined. Examination of both plant and animal mitochondria shows quite clearly that the mitochondria are surrounded by a morphological membrane; in some animal mitochondria this has been measured as 165A in thickness. Measurements which have been done of plant mitochondria suggest that the membrane may be thicker but this requires re-investigation, with the more modern techniques. Further the sectioned mitochondria examined on the electron microscope show that some are divided by internal partitions which appear to be membranes of about the same thickness as the external membrane.

If the hypothesis which we have developed to connect respiration and secretion is correct, then clearly the processes must occur in the mitochondria in which the cytochrome oxidase is located. It seems logical therefore to examine the relation of isolated mitochondria to the presence of electrolytes in the solution which surrounds them. Work of this kind has been going on in our laboratory during the past year. We have taken mitochondria from one concentration of salt to another keeping the osmotic pressure constant with sucrose. When this is done, mitochondria adjust themselves to the new concentration of salt quite rapidly but they do not reach equality of concentration with the external solution Potassium, sodium and chloride ions are retained in higher concentration in the mitochondria than in the external solution. This suggests that the accumulation mechanism is located, as our hypothesis would require, in the mitochondria themselves. It is not yet clear how this accumulation of ions is related to the metabolism of the mitochondria. But it seems certain that judging from the time it takes for the mitochondria to adjust themselves to a new concentration in the surrounding solution, the membrane or membranes of the article offer some resistance to the movement of the ions. We have calculated that if this resistance is in the external membrane which is about 200A in thickness, this membrane would be about 10 million times more resistant to the movement of salt than would water. Hence it is possible that the mitochondria are responsible, not only for the generation of the positive and negative charges but also for a system in which the hydrogen ions can be kept apart from the hydroxyl ions in secretion of HCl and could be kept apart from them in salt accumulation until the hydrogen ions have swapped for cations and the hydroxyls have swapped for anions. The properties of the mitochondrion however are still under investigation. There is a big gap in size between the mitochondrion and structures at the molecular level. Perhaps this is best summarised by saying that if the whole mitochondrion were filled with protein molecules of average molecular weight 50,000, there would be 1,000,000 such molecules.

If our hypothesis is correct in suggesting that the mitochondrion is the site of separation of positive and negative charges we must account for the observed phenomena that the HCl passes from the secreting cell into the

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stomach and, in accumulation, that the salts are accumulated in the vacuole or pass from the cytoplasm to the vacuole. What is the behaviour of mitochondria in secreting and accumulating cells? In the cells of the mucosa during secretion the mitochondria pass to one side of the cell where they lie in considerable quantity and swell to unusual size, referred to by the early cytologists as “secretory granules” In such a position the mitochondria might well have an organization which leaves the hydrogen ions on one side and passes the electrons to oxygen on the blood stream side of the cell. In plant cells which accumulate, the behaviour of mitochondria is different. One of the most characteristic processes of plant cells is that of protoplasmic streaming or cyclosis as it is sometimes called. In the course of streaming the mitochondria are swept along and passed to different regions of the cell. It seems not improbable that if the mitochondria, as a result of their metabolism have become, so to speak, charged with ions towards the outside of the cell, they may alter their ionic composition as they change their metabolic rate in different regions On the inside of the plant cytoplasm surrounding the vacuole their is a membrane which exercises a considerable resistance to diffusion of ions. If the mitochondria lose their contents when in contact with this membrane then the ions would be trapped in a region from which they could escape only slowly, but I am afraid that this is still in the stage of being purely speculative.

I have presented you with a picture in which the driving force for the separation of positive and negative charges is the respiration of the cell. The total energy necessary to accumulate salts in plant cells is only about 0.1% of the energy liberated in the salt respiration simultaneously. This is a very small part of the many things with which respiration is concerned but it has had the profound effect of making the control of composition within the cell, and, where necessary, within an organ an evolutionary tool on which the structure and function of many organisms depend. This is a problem of fundamental interest to biology. It is not solved but we have some suggestions as to how these minute cells may maintain their contents against the drift to equilibrium in an 80% aqueous medium. However difficult you may have found this technical paper which I was asked to give, I hope that at least you have seen how the struggle against equilibrium in these particular cells raises fascinating problems of physical chemistry in biology.

I have the responsibility of supervising applied research on the storage of fruits and vegetables, and of conducting plant physiological work fundamental to our understanding of the biological problems raised by storage. Since storage involves maintaining cell contents in a healthy condition for as long as possible, I need offer no excuse for our investigations on organization within the plant cell. More than that, our understanding of the biochemical and physico-chemical organization of cells in their various forms is as important and as central to biology as is the structure of atoms to chemistry and physics.

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