Linus Pauling (1964) - The Structure of Molecules in Relation to Medical Problems

I am happy to be here at the Lindau meeting, for the first time for me and I am pleased to be able to speak about molecular structure in relation to medicine. This is a small part of a great subject, chemistry, in relation to medicine, a subject that I shall not attempt of course to cover as a whole. I shall talk about molecular diseases and a little bit about a new theory, rather new theory of general anaesthesia. Now in a sense of course almost all diseases are molecular, in that the human body is made up of molecules and effectors of disease or viruses, rickettsia bacteria are made up of molecules. There are some diseases I think that we could say clearly are not molecular diseases if as the result of an accident, a man loses a large part of his brain tissue. He has mental disease. It is macroscopic in nature, the molecules that remain to him are no different from those that he had before. Or even if his brain is injured by anoxia, so that damage is done. Or some other organ is injured by anoxia although we might say that the injury is chemical in nature, I would not say that the disease that results is a molecular disease. Some years ago, nearly 20 years ago now the idea that the disease could be a molecular disease occurred to me. I should say that today I am not going to try to cover the whole subject of molecular structure in relation to disease, but just those aspects of the subject with which I have some special acquaintance or in which I have a special interest. Some nearly 20 years ago when I was serving as a member of a medical research committee investigating the support of medical research by the United States government, one of the members of the committee talked about the disease sickle-cell anaemia. This is a disease, a hereditary disease in which the red cells in the blood are twisted out of shape and as a consequence of the deformation of the red cells in the blood, the patient has a serious anaemia. His red cells are destroyed so rapidly by the spleen that he is not able to manufacture new ones rapidly enough to keep him in good health. And also these deformed red cells clog up the capillaries in crisis of the disease. So that the blood ceases to flow through some organ and the organ is damaged by anoxia. The statement that was made that was most interesting to me and that immediately brought a response from me was that the red cells are twisted out of shape in the veins, in the venous circulation, but resume their normal form in the arterial circulation. Now I have liked the haemoglobin molecule for a long time. In 1934 I began to work on the haemoglobin molecule and it seemed clear to me that there was a high probability that it was the haemoglobin molecule that was involved in this difference in behaviour of the red cells of these patients. So far as the red cells in the venous blood and in the arterial blood. The haemoglobin molecule contains about 10,000 atoms, 4 of them are iron atoms. In the lungs an oxygen molecule can attach itself to each of the 4 iron atoms. The blood charged with oxygen in this way circulates out to the tissues and the oxygen molecules are given up. The difference between venous blood, the principle difference between venous blood and arterial blood is that venous blood contains molecules of haemoglobin without oxygen attached to the iron. And arterial blood contains molecules of oxyhaemoglobin. Well the suggestion then that it is the haemoglobin that is involved brought a further idea that the patients with this disease manufacture an abnormal sort of haemoglobin that is self-complementary so that the molecules clamp on to one other to form long rods which line up side by side as a crystal or perhaps tactoid liquid crystal of haemoglobin which as it grows longer and longer twists the red cell out of shape and leads to the manifestations of the disease. When the iron atoms are oxygenated the self-complementarianist may be destroyed in such a way that the crystals go back into solution and the cells resume their normal shapes. When I return home to Pasadena a few months later, a man who had, a young man, Harvey Itano who had received his MD degree came to work with me and I asked him to examine the haemoglobin of patients with this disease and to compare the haemoglobin of these patients with haemoglobin of normal individuals. For 3 years he made comparisons of haemoglobins from these 2 sources. And always with the same result in every experiment that he carried out. Well with a substance so complicated that its molecules contain 10,000 atoms, the identity, apparent identity of certain properties is no assurance that the molecules are completely identical in structure. Finally he carried out one experiment, he and 2 other young men, Doctors Singer and Wells then working with him, carried out one experiment, electrophoresis experiment in which the 2 samples of haemoglobin behaved differently. That was enough to show that they are different, this one experiment. With such a complicated substance any number of experiments in which they behaved the same would not be proof of identity. Many other abnormal human haemoglobins have been discovered since 1949 and many abnormal or different haemoglobins manufactured by other animal species, several haemoglobins by the same species of animal, under the control of course of genes. I believe that I shall continue now with the slides. Here we have, and perhaps it is, I can say as a tribute to Professor Meccarr (inaudible 8.38) as well as to my fellow Californians, both now dead, Professors. They were young men then, Doctors Latimer and Rodebush who in 1920 discovered the hydrogen bond that I start out with a picture of a molecule containing hydrogen bonds. The dimer of acetic acid, the hydrogen bond is of such great importance in the molecular structure of the human body that I have chosen to start with this representation. The 2 oxygen atoms bonded by a hydrogen atom are 2.79 Angstrom apart. And the bonds, the 2 bonds together amount to about 14 kilocalories per mole. The next slide. This is an old drawing going back some 15 years indicating that in the polypeptide chains of proteins stable structures will be those in which the polypeptide chain is folded in such a way that hydrogen bonds are formed. At the time that this drawing was made, no configuration of polypeptide chains had yet been discovered, none of the configurations in which the polypeptide chains occur in nature. The next slide. This shows the folding of a polypeptide chain into the Alpha helix. The peptide groups are plainer because of the partial double bond character of the carbon nitrogen bonds here stealing double bond character from the carbonyl group of the amide. There is essential freedom of rotation around the single bonds to the Alpha carbon atoms. The folding is done in such a way that each hydrogen atom attached to hydrogen is able to form a hydrogen bond with the carbonyl oxygen in the peptide group 3 removed from it along the chain. There are 3.6 amino acid residues per turn of the helix. The next slide please. And the pitch, the distance along the helical axis per residue is 1.49 angstrom. The dimensions of the Alpha helix have been very well verified by experiment for the synthetic polypeptides with the Alpha helix structure for Alpha keratin, fibrous proteins, hair, horn, fingernail, porcupine quill and so on, and also through the work of Kendrew for the polypeptide chain of the globular protein myoglobin. Myoglobin and Professor Kendrew will I hope speak in detail about his great feat in making an essentially complete structure determination of the myoglobin molecule. Myoglobin contains 8 Alpha helix segments, one of which is shown here. The iron atom is up in the upper right hand corner surrounded by the atoms of the porphyrin group that constitute with the iron atom the haem group where the oxygen is attached. Now here we have a patient with the disease sickle-cell anaemia. This disease seemed to be a disease of the red cell and then turned out to be a disease of the haemoglobin molecule. The next slide. In the oxygenated blood of these patients, the red cells have a normal appearance when seen through the microscope. In the deoxygenated blood, in the veins they are sickled, the red cells are sickled. When this investigation was first carried out showing that the patients produce an abnormal haemoglobin, the parents were, of a patient were studied. It was found that the father contained in his red cells a mixture, 50% of the haemoglobin was normal, 50% abnormal, similarly for the mother. Here we have a paper chromatographic study. The diagram on the far right is the best, it corresponds to 4 hours of electrophoresis. At the bottom is a haemoglobin from a normal individual, it migrates rapidly, it has a large negative electric charge. Directly above it is the haemoglobin from the father or mother of a sickle-cell patient. Here there are 2 haemoglobins, a normal haemoglobin and sickle-cell haemoglobin. The sickle-cell haemoglobin migrates at a rate showing that it differs in its electric charge by 2 electronic units. It has 2 fewer negative electric charges than the normal haemoglobin has. Doctor Itano brought into the laboratory a sample of blood from another person. When he investigated it, it was found that this person had sickle-cell haemoglobin in his red cells and another haemoglobin still more abnormal than the sickle-cell haemoglobin, with 4 units of charge difference from normal haemoglobin. The one parent of this interesting individual was a sickle-cell heterozygote, the other was a heterozygote in this new abnormal haemoglobin, manufacturing both normal haemoglobin and this new abnormal haemoglobin. In the theory of heredity, Mendelian heredity, one would expect that parents of this nature, One quarter of the children would inherit the abnormal gene of the father and also the abnormal gene of the mother and would then have a double abnormality each present in single dose. This person had the disease of a new kind, a disease involving the inheritance of 2 different abnormal genes which separately do not produce any serious disease. But they cooperate with one another to produce a new type of anaemia, the disease is called sickle-cell haemoglobin C disease. This haemoglobin was named haemoglobin C. Many other haemoglobins have since been discovered, haemoglobin D, E, G, H and so on, scores of human abnormal haemoglobins are now known. So many that I can’t keep up. Next slide please. A few of them are indicated here. Along the diagonal of this matrix we have some of the homozygotes, the sickle-cell patients with 2 sickle-cell genes, haemoglobin C patients with 2 haemoglobin C genes and so on. At the top are the carriers of the genes in single dose, they in general do not have serious diseases. Then we have on the diagonal some of the complex diseases involving the inheritance of 2 different abnormal genes and the manufacture of 2 abnormal haemoglobins. The next slide. Doctor Schroeder in our laboratories and his collaborators using the method of Sanger were able to show that there are 2 kinds of polypeptide chains in the normal haemoglobin molecule. One chain contains 141 amino acid residues. It is called the Alpha chain, it begins with a residue of valine and continues leucine, serine, proline, alanine, asparagine and so on. The Beta chain, the other chain begins valine, histidine, leucine, threonine, proline, glutamic acid and continues on. An English investigator, I’ll think of his name in a moment, Vernon Ingram developed a technique of two-dimensional paper electrophoresis chromatography and the splitting of haemoglobin into several simple peptides and was able to show that the abnormality and sickle-cell haemoglobin is in the Beta chain. He and Doctor Schroeder tied it down to the 6th position in the Beta chain where valine replaces glutamate. The glutamate residue carries a negative electric charge. The carboxylate group is ionised and valine has a hydrocarbon side chain with no electric charge. Consequently one negative electric charge is lost from this substitution. The next slide please. There are 2 Beta chains in the sickle-cell haemoglobin, 2 Alpha chains and 2 Beta chains, just as in normal haemoglobin. But the Beta chains are changed by the substitution in the 6th position in the chain, giving them a difference in electric charge of 2 units and a difference in molecular structure such as to produce the complementariness and insolubility characteristic of sickle-cell haemoglobin. We do not yet, despite the work of Perutz in Cambridge, we do not yet know the structure of the haemoglobin molecule well enough to be able to explain in terms of structure the formation of the tactoids by deoxygenated haemoglobin S. But we can expect that this will occur soon. Here, there is a symbol given for haemoglobin F, the letter F stands for foetal, haemoglobin F is the haemoglobin that is manufactured by the foetus. It contains 2 normal Alpha chains, resembling the adult and 2 Gamma chains which are rather different from the Beta chains. At about the time of birth of an infant the infant begins to manufacture Beta chains whereas earlier in life, in prenatal life he was manufacturing Gamma chains. Next slide please. This is the technique that Ingram used of hydrolysing a protein with tripsin, the haemoglobin to produce about 26 peptides, each with 10, 12, 14 amino acid residues in it. Separating on the paper by electrophoresis, in this direction and by chromatography vertically. Only one of the 26 peptides is different in sickle-cell haemoglobin from normal haemoglobin. It is represented by this spot which has moved to this position. And when this peptide was investigated it was found to be the first peptide in the Beta chain and to have glutamate replaced by valine. The next slide. Here we have results indicated for some other abnormalities of human haemoglobin molecules involving the Beta chain, many abnormal haemoglobin, human haemoglobins are known in which the Alpha chain is abnormal, they are not shown here. There are 146 amino acid residues in the human haemoglobin Beta chain, they are all known, only 31 are indicated here in the first line across here. In the case of haemoglobin S, sickle-cell haemoglobin as I have mentioned in the 6th position, glutamate is replaced by valine. With haemoglobin C glutamate is replaced by lysine. Now lysine has an amino group attached to the Delta carbon atom of the side chain and it becomes an ammonium ion group, physiological pH. So that the lysine side chains carries a positive electric charge, the glutamate a negative electric charge with 2 Beta chains in the molecule, this means a difference of 4 units of electric charge between haemoglobin C and normal haemoglobin. Haemoglobin G has a substitution in the 7th position, haemoglobin E a substitution in the 26th position, haemoglobin A2 a substitution in the 22nd, it is accident that all of the substitutions that are indicated here involve replacing a glutamic acid residue by some other residue. Other kinds of substitutions are known. In each case as for example haemoglobin S, sickle-cell haemoglobin, there is only 1 amino acid residue changed. All of the other 140 are exactly the same as in the normal adult human haemoglobin. No variant of human haemoglobin has been discovered in which either the Alpha chain or the Beta chain differs from normal by more than 1 amino acid residue. The next slide shows the geographical distribution of the gene for sickling, Central Africa, Madagascar and then this is Atlantis I think down here. Then various isolated occurrences reported. And of course in the United States, in Sicily, Southern Italy, Greece, these regions over in Portugal there are numbers of people who carry the sickle-cell gene. One can ask why this gene has spread so widely among the human population, there must be some advantage to carrying the gene in order for a mutation to begin to spread. The answer was suggested by a British physician, Doctor Breen (inaudible 24.35) who noticed that there were more sicklers, people’s whose red cell sickled in malarial regions in Africa than in non malarial regions. Then a young physician, Doctor Anthony Allison carried out a crucial experiment in Kenya, he got 30 healthy adult Africans, male, who were shown by skin tests not to have developed any immunity to malaria. When they were inoculated with malignant subtertian malaria, 15 of these who were normal people, so far as their haemoglobin goes, became ill. But of those who had one sickle-cell gene, the heterozygotes in the sickle-cell gene only 2 came down with malaria. There was a great degree of protection against malaria by a single gene. Their red cells contain a 50/50 mixture of normal haemoglobin and sickle-cell haemoglobin. And this provides them with protection against malaria. There is a molecular mechanism of course. Ordinarily the red cells of these individuals, the heterozygotes do not sickle in the venous circulation. But if the blood is completely deoxygenated then the red cells are twisted out of shape. The crystal forms even though the haemoglobin is diluted with an equal amount of normal haemoglobin. Well the malarial parasite lives inside the red cell and the parasite has a high metabolic rate. He uses up the oxygen inside the red cell, so that the partial pressure of oxygen becomes so low that the haemoglobin crystallises, twists the red cell out of shape and squashes the parasite to death. So we have a molecular explanation, not only of the lethal manifestations of the abnormal haemoglobin in the homozygotes but also of the protection against malaria that is provided to the heterozygotes. The next slide. This shows the incidence of haemoglobin C, high incidence in northern Ghana, low in southern Ghana and then still smaller along the coast here. It seems likely that the haemoglobin C mutation occurred only a short time ago, perhaps 1,000 years ago whereas the mutation producing the sickle-cell haemoglobin may have occurred 5,000 or 10,000 years ago and then have spread over Africa. The next slide. Now I want to discuss another type of disease, also related to haemoglobin and I begin by showing again the structure of myoglobin. I point out the iron atom and a group here that is rather close by the iron atom. This group is a histidine residue, it is in the 58th position of the Beta chain or the 63rd position of the Alpha chain. This group is I believe responsible for the retaining of the iron atom in the ferrous state. Haemoglobin can also be called ferro-haemoglobin. The iron is bipositive, bivalent. Sometimes haemoglobin is oxidised to the tripositive state, the iron becomes ferric rather than ferrous. And this ferri-haemoglobin also called met-haemoglobin does not have the power of combining reversibly with oxygen so that the power of transferring oxygen from the lungs to the tissues is lost. Now ordinarily ferrous compounds are easily oxidised to the ferric state. This residue of histidine has an imidazolium ring in its side chain. The imidazolium ring at physiological pH adds a proton and assumes a positive charge. I believe that this positive charge in the neighbourhood of the iron atom stabilises the ferrous state by repelling the additional positive charge that would be added to the iron atom to convert it from iron plus 2 to iron plus 3. And there is good evidence for this now through the investigation of the haemoglobins of certain people who have a disease in which half of the iron atoms, 2 of the iron atoms in their haemoglobin molecules are easily converted and naturally converted to the tripositive state, to ferric iron. Doctor Gerald has been responsible for much of the investigation of the haemoglobins of these patients with the disease met-haemoglobinemia or ferri-haemoglobinemia. The next slide. Here we have the group of some 60 atoms that is called the haem group with the iron atom at its centre bonded to 4 nitrogen atoms of 4 pyrrole rings and so on in the way that Professor Fischer, Hans Fischer showed 60 years ago. Next slide. Without having knowledge of the dimensions of course that we have now. Here we have the haem group with the side chain of histidine, the imidazolium cation carrying a positive charge that stabilises the ferrous state of the iron. This you find in normal persons. Next slide. Now here is a type of variant haemoglobin, haemoglobin Zürich in which in the Beta chain, Tyrosine with a para hydroxyl benzene ring does not pick up a proton that does not carry a positive charge. The iron atom easily oxidises to the ferric state and the patient has the disease ferri-haemoglobinemia. The next slide shows another abnormal haemoglobin in which the same histidine residue is replaced by arginine. Now arginine has a guanidine group in its side chain that picks up a proton to produce the guanidinium cation of the positive charge stabilises the iron atom, it does not oxidise to the tripositive state. And these people even though they carry an abnormal haemoglobin with the abnormality in this critical position do not have the disease ferri-haemoglobinemia. The next slide. Here we have a few of the abnormal haemoglobins related to this state. Here is a Beta in which there, well let us go to this one, Boston has tyrosine in the 58th position of the Alpha chain, Emory has tyrosine in the 62nd position of the Beta chain, they both lead to ferri-haemoglobinemia. Zürich has arginine in this position for 62nd in the Beta chain but without producing ferri-haemoglobinemia. And interesting abnormal haemoglobin is Milwaukie which has glutamic acid in position 4 removed from histidine. The Alpha helix has nearly 4 residues per turn of the helix. And this residue of glutamate is near the iron atom too. Normally there is valine in this position which does not carry a charge. The negative charge of glutamate apparently attracts an extra positive charge to the iron atom which becomes a ferric iron atom and produces ferri-haemoglobinemia. Here we have a disease then for which there is a simple detailed chemical explanation of the manifestations of the disease. It will be hard to go beyond this, deeper than we have gone now with this group of diseases in understanding disease on a molecular basis. The next slide please. I should like to mention some evolutionary considerations. These involve studies carried out in our laboratories, mainly by Doctor Emile Zuckerkandl from France with the Centre National de Recherche Scientifique. We have here the peptide patterns using the method of Ingram for human haemoglobin, fish haemoglobin, shark, down here, hog fish, echiuroid worm. It is clear that there are great differences in the haemoglobins. In fact the differences look to be greater than they actually are because there are great similarities too, even between human and fish haemoglobins. The next slide. We see now a comparison of human, cow and pig. And it is evident that the patterns are somewhat similar. The haemoglobin structures are rather similar. The next slide shows human, chimpanzee, gorilla, orang-utan, rhesus monkey. If we compare human and gorilla or human and chimpanzee it is nearly impossible to find a difference. And a more detailed investigation shows that with human, the Alpha chain of the gorilla differs from the Alpha chain of the human by 2 amino acid residues out of 141. The other 139 are the same and in the same positions. The Beta chain of human and gorilla, the Beta chains differ by 1 amino acid residue only. In the case of chimpanzee, the Beta chain of chimpanzee differs from that of human by 1 amino acid residue. And it is identical with a type of human Beta chain, human haemoglobin called Norfolk haemoglobin, probably an independent mutation. However in the case of the Beta chain of the people in Norfolk who have this similarity, identity with the chimpanzee Beta chain. With rhesus monkey instead of 1 or 2 differences there are 6 or 8 differences. With horse there are 18 differences, 18 amino acid residues out of 141 or 146 that are different. The next slide. Doctor Zuckerkandl and I thought that we would try to make some statements about the process of evolution, we took horse and human haemoglobins and assumed that the line leading to humans and horses, these lines separated 130 million years ago, that’s why the brackets are here, this is the starting point. And then we assumed that there is a constant rate of mutation, evolutionarily effective mutations. With this assumption the gorilla Alpha chain corresponds to 14 million years ago, the Beta chain to 7, the average is about 11 million years ago. I don’t have comparison of human and rhesus monkey band but it would come out about 40 million years ago. And this at once answers a question that the students of evolution have asked, at what stage did the monkeys of different kinds and the anthropoid apes and man, at what stage did these lines separate from one another. The monkeys separated off from the common ancestor of gorillas and human beings about 40 million years ago. The gorillas and human beings separated roughly 10 million years ago. I like this, 260 million years ago was when human foetus separated from human adult. Well of course there weren’t humans then, it was when foetuses separated from the adult. A human foetus is more like a horse foetus in its haemoglobin, the Beta chain or Gamma chain of its haemoglobin than like an adult human being. In a sense so far as the Gamma chains, Beta chains go, the foetuses of mammals are more closely related to one another than they are to their corresponding adults. Now the Alpha chain and the Beta chain have about 78 differences and some 60 identities, corresponding to about 600 million years ago. The identities are so numerous that we can be sure that they originally represented a single gene, a single chain. Next slide please. And we have attempted to determine the nature of the single polypeptide chain of the haemoglobin manufactured by the ancestor of all vertebrates some 600 million years ago. Here we have just 4 different chains indicated and as we move along we see that in this position all 4 have lysine. In this position 3 of them have leucine and the fourth has phenylalanine. It looks as though the foetal gene has undergone a late mutation. After the separation of the genes for Beta and Gamma, gene duplication followed by independent mutation, the mutation occurred in the Gamma gene. The next slide shows our present knowledge about the amino acid sequence in the polypeptide chain for haemoglobin manufactured by the common ancestor of all vertebrates some 600 million years ago. There are some uncertainties, great uncertainties, about half the positions are not filled. Here there’s one position not filled, another one with no knowledge, another one, in a few years more I’m sure that all of these positions will be filled. It might then be possible to synthesise the polypeptide chain, add the haem to it, determine the oxygen combining power of this primeval form of haemoglobin. And in that way make a decision, reach a conclusion about the partial pressure of oxygen in the atmosphere on earth 600 million years ago. The next slide. Now I shall talk briefly about another application of molecular structure to a problem that we can call a medical problem, the problem of the nature of general anaesthesia. There has not been any satisfactory theory, perhaps there still is no really satisfactory theory but there has hardly been a theory of general anaesthesia until the theory that I shall describe was developed about 4 years ago. I published this in June of 1961 and a few months later a young chemist, Doctor Stanley Miller, Professor Stanley Miller in the University of California in San Diego published essentially the same theory. Quite different words and different calculations but I think that it’s the same theory. I have here a drawing of the structure of ordinary ice. Each water molecule forms 4 hydrogen bonds with its 4 neighbours, it looks as though there are holes in this crystal but these holds are really not very large. No molecules except helium and hydrogen I think could fit into these holes up here. Now the next slide shows another view of ordinary ice looking down the hexagonal axis, the atoms are really much larger so that the holes are small. Next slide. Here we have an aggregate of 20 water molecules forming 30 hydrogen bonds with one another. The bond angles within the pentagonal dodecahedron are 108 degrees so that no bond angles strain from the tetrahedral angle 109 degrees and a half is involved. Investigation of hydrate crystals showed that these pentagonal dodecahedra of 20 water molecules occur in many of them. The next slide. This is the structure, basic structure of chlorine hydrate, methane hydrate, xenon hydrate. I was especially interested, this structure was determined by Doctor Marsh in our laboratory 12 years ago, I was especially interested in xenon hydrate, the fact that xenon formed crystals with this framework. The xenon atoms occupying positions at the centres of the polyhedra. There are 6 extra water molecules in addition to the 40 of the 2 dodecahedra. The next slide shows that there are not only the dodecahedra but also tetrakaidecahedra in the centres of which somewhat large molecules such as metal chloride or cyclopropane or chlorine can fit. These crystals with their rather open framework, hydrogen bonded framework of water molecules are stabilised by the Van der Waals interaction of the xenon molecules or cyclopropane molecules or other molecules that occupy the cavities with the water molecules. The next slide. This is a larger cavity formed by 28 water molecules. It has 4 hexagonal faces and 12 pentagonal faces. And it is large enough to permit a molecule of chloroform, CHCL3 to fit inside it. In the chloroform hydrate crystal, CHCL3 17H2O, there is one of these polyhedra for every 2 dodecahedra. If xenon is present the melting point, the decomposition point of the crystal is raised by14 degrees centigrade, from 2 degrees to 16 degrees centigrade because of the Van der Waals interaction of the xenon molecules with the neighbouring molecules. There’s a cooperation then in this crystal, 2 xenon CHCL3 17H2O, a cooperative effect involving the xenon in stabilising the crystal. The next slide. This is a still larger opening in a hydrogen bonded framework in which there is a tetra iso-amyl-ammonium ion and a fluoride ion also present. I was reading a manuscript describing a crystal of this sort, not yet published paper sent to me by Professor Jeffrey back in 1959, April 1959 when I thought to myself I understand the mechanism of anaesthesia, general anaesthesia. Here we have an ion, something like the side chain of a lysine residue in a protein, perhaps in the brain. These electrically charged side chains and some ions interact with the water molecules to form small crystals of a hydrate. And in the presence of an anaesthetic agent which can fill other cavities, xenon for example which serves as a good general anaesthetic, it isn’t used because it’s so expensive. Xenon molecules, xenon atoms could occupy some of the smaller cavities and stabilise the crystal which however would also involve some ions from the solution and some of the side chain groups of proteins. This would trap the electrically charged side chains and ions which normally oscillate back and forth contributing to the electric oscillations in the brain that constitute consciousness and ephemeral memory. And by decreasing the amplitude of the oscillations, electrical oscillations would lead to unconsciousness. Then as the anaesthetic agent is allowed to leave the body through the lungs, the crystals would become unstable and would decompose again and consciousness would be regained. If this theory were right then it should be possible to anesthetise people by cold, just by cooling the brain. And of course I discovered that it is possible to produce anaesthesia just by cold. At 30 degrees centigrade mild anaesthesia is produced, at 26 degrees deep anaesthesia. Also, next slide please, also if this theory is right, there should be a close relation between the Van der Waals forces, between the anaesthetic molecules and other molecules and the anaesthetic activity. Now here I have plotted the logarithm of the equilibrium partial pressure of the hydrate crystals of various anaesthetic agents. Against the molecular polarisability expressed here in cubic centimetres per mole. The high pressure, nearly 100,000 millimetres of mercury is required to produce argon hydrate crystals, methane, krypton and so on, xenon down here, metal chloride, ethyl bromide, chloroform, carbon tetrachloride. We have this curve. Over here is the curve similarly plotted against the molecular polarisability. The logarithm of the narcotising or anaesthetising partial pressure for mice at 37 degrees centigrade. And the curve has the same smooth character. Next slide. Here I have plotted the anaesthetising partial pressure, logarithm of it against the logarithm of the equilibrium partial pressure of the hydrate crystals. There is a reasonably good linear relationship which goes over a great range of pressures. Here we have 10, 100, 1,000, some 3,000 fold range in anaesthetising partial pressures between chloroform and argon as shown on this curve. This is then some indication that it is the molecular polarisability that is responsible for the anaesthetic activity, not necessarily the formation of hydrate micro crystals. There might be some other way in which this molecular property could be acting. But I think that the hydrate micro crystal idea is a good one. The next slide. Here are some experiments not yet published carried out with goldfish in which, here we have the temperature, the reciprocal temperature, temperatures above running from zero degrees over here, 1.6 degrees to 35 degrees. For these several anaesthetic agents the temperature coefficient of the anesthetising partial pressure, the logarithm is shown here plotted against reciprocal of the temperature corresponds over a wide range to nearly constant entropy of reaction with nearly the same value for the different anaesthetic agents. Then there is a rapid drop, the gold fish are anesthetised at 1.6 degrees centigrade even in the absence of the anaesthetic agent. This sort of catastrophe of course indicates a cooperative phenomenon such as crystallisation. A phenomenon in which a large number of molecules take part, a change in phase. And I think that this is a good indication that something that we might call microcrystal formation is taking place. The next slide please. This is another representation of the crystal of xenon hydrate in which there is the hydrogen bonded framework of water molecules with a xenon molecule, monatomic molecule occupying each of the dodecahedral and tetrakaidecahedral cavities. I am pleased that a reasonable theory, lights please. I am pleased that it is possible to propose an explanation of the extraordinary property of xenon, highly un-reactive substance which surely does not enter into ordinary chemical reactions in the human body of producing anaesthesia when it is inhaled. I think that it is possible to understand the molecular structure of the human body, to understand physiological phenomena, even psychic phenomena, I believe that it will be possible to get a penetrating and deep understanding of the nature of mental disease in the course of time. Such as to permit great progress to be made in the control and treatment of mental disease which is one of the great scourges of course in the world today and the cause of a tremendous amount of human suffering. We are just entering now on the period of development of molecular biology and medicine. The ideas are necessarily rather crude ones that have been proposed so far but I think that we can have great hope for the future. Thank you.

Linus Pauling (1964)

The Structure of Molecules in Relation to Medical Problems

Linus Pauling (1964)

The Structure of Molecules in Relation to Medical Problems

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Linus Pauling was an incredibly productive and versatile scientist. His work comprised fundamental problems from the fields of chemistry, physics and medicine. At the same time, his commitment ranged far beyond the borders of science. To this day, he is the only person who received two Nobel Prizes without co-recipients: the 1954 Nobel Prize in Chemistry for his work on the nature of the chemical bond and chemical structure as well as the 1962 Nobel Peace Prize for his fight against nuclear weapons. Pauling used his 1964 Lindau lecture to talk about some of his most recent research at the time, which he summarized under the umbrella of “chemistry in relation to medicine”. The first part of this talk concerns two diseases relating to haemoglobin, the protein responsible for oxygen transport in blood. In the second part, he tries to answer some seemingly trivial questions: how is it possible that xenon, a noble gas completely devoid of any physiologically relevant chemical reactivity, can act as a general anaesthetic? And is there a common mode of action of general anaesthetics? That these questions are indeed tricky ones can be seen from the fact that even today (2013), almost fifty years after Pauling’s talk, a satisfactorily comprehensive theory of general anaesthesia is still not available. Although general anaesthetics have been used for more than 150 years, we just do not know how they work entirely. In a couple of papers published from 1961 to 1964 [1-2], as well as in the present lecture, Pauling proposed that the only property common to all known anaesthetics is their effect on water crystallization and that the loss of consciousness achieved by anaesthesia is indeed due to the spontaneous formation of water microcrystals in the brain. While water itself naturally does not crystallize at the temperature of the human body, hydrates of certain other molecules possibly could, Pauling says. This is due to the fact that water crystals contain relatively large cavities, which are usually unoccupied (this is the reason for ice floating on top of liquid water). However, certain small molecules, like xenon, can occupy such cavities via the formation of hydrates and thus stabilize the crystal even at elevated temperatures. This stabilization is due to the so-called London dispersion force and does not require any chemical reaction to take place. In his “hydrate microcrystal theory of general anaesthesia” Pauling now proposes that such spontaneously formed microcrystals might interfere with the motion of ions or electrochemically charged protein side chains, which are essential to consciousness and short-term memory. He presents some convincing correlations between the polarizability of molecules (which determines the strength of the London dispersion forces) and its anaesthetic potential. However, in a 1964 paper [2], he also points out that his general theory is not so general after all: the effect of certain anaesthetics like the barbiturates and diethyl ether cannot be explained by it. Today, it is accepted that general anaesthetics do not act by one single mechanism but rather affect a number of specific protein targets [3], both in the brain and the spinal cord. A comprehensive explanation by a single mechanism thus appears unrealistic.The present lecture is the first of four lectures Pauling gave in Lindau. In 1977 and 1981 he should return to talk about his controversial suggestion that high vitamin C dosages could protect from cancer. His last Lindau lecture, held in 1983, dealt with the structure of transition metal compounds, whereas extended parts of it are dedicated to the issues of nuclear weapons and peace. David Siegel[1] L. Pauling, Science 134 (1961) 15.[2] L. Pauling, Anesthesia & Analgesia 43 (1964) 1.[3] N.P. Franks, Nature Reviews Neuroscience 9 (2008) 370.

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