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Mini‐series: Significant contributions to biological chemistry over the past 125 years: Biochemistry in the United States in the first half of the twentieth century

2002; Wiley; Volume: 30; Issue: 2 Linguagem: Inglês

10.1002/bmb.2002.494030020034

1539-3429

Mildred Cohn,

Various Chemistry Research Topics

To attempt to cover the significant contributions to biochemistry in the first half of the 20th century in the United States is a daunting task. My task has been made somewhat more achievable by the instruction to focus on those contributions that have most influenced current research. Furthermore, to limit the discussion to research in the United States without mentioning the interaction with science elsewhere is neither possible nor desirable, and will not be attempted. The contributions that I have chosen to highlight unavoidably reflect my personal bias. Because Howard Gest has covered biological oxidations and bioenergetics [1], that area will be omitted from my discussion. I shall limit my talk to work that was done or initiated before 1946; a new era in biochemical research began after World War II. At the beginning of the twentieth century, biochemistry was practically non-existent in the United States, being actively pursued in Europe with its center in Germany. In fact most of the productive American biochemists in the period before 1920 received training in Europe, usually Germany. For example, of the 24 founding members of the American Society of Biological Chemistry in 1906, only seven had not received training in Europe. In the first three decades of the twentieth century, biochemical research in this country was focused primarily on analysis of the components of body fluids, tissues, and excreta, i.e. clinical chemistry, and on the study of nutrition. By 1930 the leadership in both areas had moved from Germany to the United States. The development of analytical methods was led by Otto Folin of Harvard and Donald Van Slyke of the Rockefeller Institute. The leadership in nutritional research came from Thomas Osborne of the Connecticut Agricultural Experiment Station and Lafayette Mendel at Yale, followed by Conrad Elvehlem, Harry Steenbock, and Elmer McCollum at the University of Wisconsin. Other luminaries among the biochemists of that period included the first professor of Physiological Chemistry in the United States, Russell Chittenden, at the Sheffield Scientific School at Yale, beginning his tenure in 1882 and retiring in 1921. Chittenden had a broad vision of biochemistry, embracing all biology similar to what Gowland Hopkins had achieved in England, but he never succeeded in his effort to create a university department. During his tenure, he was most influential in the development of physiological chemistry throughout the country. Another noteworthy biochemist was John Abel, who spent seven years in various research laboratories in Germany, eventually receiving an M.D. degree from Strasbourg. He first isolated epinephrine and was the first to crystallize insulin when he was a professor at Johns Hopkins. Abel persuaded Christian Herter to finance the publication of the Journal of Biological Chemistry in 1905. Herter was independently wealthy, and although he was professor of pathological chemistry at the College of Physicians of Columbia University, he maintained a large and well equipped laboratory in his private home on Madison Avenue in New York for himself and several associates, the most notable being Henry Dakin. The latter, an English organic chemist, was most versatile and made contributions to oxidation-reduction processes in animals, discovered arginase and glyoxylase and synthesized adrenaline. Another outstanding biochemist of that era was Phoebus Levene at Rockefeller Institute, a bioorganic chemist in the German tradition, who specialized in the chemical composition of nucleic acids, proteins, and phospholipids. By 1920, overall metabolism in higher organisms was fairly well elucidated. Not only were the chemical identities of the starting materials and products of metabolism known, but the extent to which protein, carbohydrate, and fat could be interconverted was also known. However there was a paucity of information on intermediary metabolism, with some notable exceptions. In 1905, Franz Knoop suggested β-oxidation of fatty acids, Hopkins found lactate formation in muscle in 1907, Arthur Harden and William Young identified hexose phosphates in fermentation, and by 1911 it was known that amino acids were oxidatively deaminated and that ketonic acids then could undergo decarboxylation. No pathways had been elucidated; only fragmentary information was available until the 1920s and 1930s when the pathways of glycolysis and fermentation were worked out by Otto Meyerhof, Gustav Embden, Jacob Parnas, Otto Warburg, and Carl and Gerty Cori, a truly monumental accomplishment considering the methods available at the time. In attempts to establish intermediates in metabolic pathways, one approach had been to substitute a hydrogen with a halogen thus making the metabolite traceable. Unfortunately, in most cases the halogenated compound was not metabolized in the same way as the natural metabolite. Some progress had been made by balance experiments, that is, a compound was fed to an animal in large amounts and an abnormal amount of a related compound was sought in the urine or tissues. There are two serious limitations to this type of experiment, one is that some compounds are never produced in excess, and two, the other side of the coin, some compounds cause an increase in another compound but are not directly involved in synthesis; for example, insulin causes formation of glycogen. To quote Rudolph Schoenheimer and David Rittenberg's analogy, “in a vending machine, a penny brings forth one package of chewing gum, two pennies brings forth two packages of chewing gum; the first observation is an indication of the conversion of copper into gum, the second observation constitutes proof [2].” The introduction of isotopes to study intermediary metabolism in 1935 by Schoenheimer and Rittenberg superseded other approaches and revolutionized the study of intermediary metabolism. Schoenheimer, a German biochemist, was one of Hitler's gifts to the United States, and Rittenberg was a student of Harold Urey, the discoverer of deuterium in 1932 who subsequently developed methods of separating it in quantity. The idea of using isotopes as tracers was first introduced in 1923 in Germany by George von Hevesy who used natural radioactive thorium B, an isotope of lead, to follow the transport of lead in a plant. Later he studied the distribution of radioactive phosphate in animal tissues, but he limited his studies to inorganic compounds. With the availability of deuterium, Schoenheimer and Rittenberg began their isotopic tracer studies on fatty acid metabolism. In an early test of the method, they found that when an animal was given D2O to drink for several days, the fatty acids became labeled with deuterium at the rate shown in Fig. 1. When D2O was removed, and the animal was subsequently given normal water instead of D2O, the label disappeared at the same rate as its incorporation as shown in Fig. 1, demonstrating for the first time that a body constituent exists in a balanced state with synthesis balanced by degradation. George Popjak has stated, “These two simple curves seem to me to have been as fundamental to the development of modern biochemistry as was Einstein's equation of the energy-mass equivalence to modern physics [3].” Although I think that evaluation overstates the case, nevertheless when Schoenheimer and Rittenberg extended this concept by demonstrating with 15N and deuterium that the same holds true for proteins, it did overturn the Folin theory of protein metabolism, which had held sway for three decades. Folin thought that proteins were stable in living cells, that ingested proteins were either broken down to liberate energy or a minor fraction used to replace “wear and tear.” The impact of Schoenheimer's concept of “the dynamic state of body constituents” on contemporary ideas of regulation is the realization that one of the significant factors in regulation is the limited life span of cell constituents. In addition to introducing the concept of rapid turnover of body constituents, Schoenheimer and his associates labeled compounds with deuterium in specific positions of organic molecules to follow their conversion. Thus they prepared labeled stearic acid chemically, fed it to mice for 12 days, killed the mice, and analyzed the isolated fatty acids for deuterium. The saturated C16 palmitic acid and the monounsaturated C18 oleic acid carried the label. When the oleic acid formed in this experiment was fed to mice, stearic acid was labeled. Thus it was established that three principal fatty acids were interconvertible in the animal body even when adequate unlabeled fat is available in the diet. With the enrichment of the 15N isotope by Urey, Schoenheimer and his associates extended their tracer studies to the study of the detailed pathways of synthesis and breakdown of labeled amino acids. The importance of transamination was demonstrated with recognition of the special role of glutamic and aspartic acids. The fate of labeled arginine agreed with Hans Krebs formulation of urea synthesis, and the long studied problem of creatine synthesis was solved when Konrad Bloch showed that arginine transferred its amidino group to glycine to form guanidinoacetic acid. This was followed by the experiments of Vincent du Vigneaud who showed with deuterium-labeled methyl of methionine that transmethylation occurred from methionine to creatine and to other compounds; these findings led eventually to the discovery by Guilio Cantoni in 1953 of the key intermediate S-adenosylmethionine. After the untimely death of Schoenheimer in 1941, his associates and students continued to use tracer methods to elucidate biosynthetic pathways well into the second half of the twentieth century, notably David Shemin's work on porphyrins, Bloch's on cholesterol, and Sarah Ratner's on urea synthesis. In the current era of regulation and signal transduction, we tend to forget that knowledge of metabolic pathways is necessary before we can study their regulation. The use of isotopes spread rapidly throughout biochemistry with the availability of 14C, which was much easier to measure than stable isotopes. 14C, discovered by Samuel Ruben and Martin Kamen in 1940 and unlike the earlier radioactive 11C, which had limited usefulness because of its 20-min half-life, has a half-life greater than 5000 years. It became available in 1945 from nuclear reactors (“atomic piles”) as did commercial counting instruments, and thousands of research publications have resulted from its use. There has been a rebirth of interest in the stable isotopes 2H, 13C and 15N in recent years, because the magnetic properties of their nuclei make them uniquely effective in NMR structural studies, particularly of macromolecules. Another area of research that advanced significantly from work in this country, both experimental and conceptual, is the structure and function of proteins. The chemical nature of proteins was racked with controversy in the nineteenth century and continued to be in the first three decades of the twentieth century. One belief favored by physical chemists was that proteins are not single molecules but colloidal in nature, that is, a relatively large number of single molecules combine to form “net-like, sponge-like masses” to produce those molecular groups that resist diffusion, the defining characteristic property of colloids. A corollary to this formulation is that the protein of living tissue must have an entirely different structure from that of dead tissue. Although Chittenden and many others apparently accepted these ideas, the European organic chemists did not. After considerable controversy, by the mid-thirties, it was accepted that proteins are discrete molecules. It was also thought that there were only several classes of proteins and that all proteins of one class, e.g. caseins from various sources, had the same chemical composition. The difficulty of determining composition lay in the lack of good analytical methods. Of the 20 amino acids in proteins, there were satisfactory quantitative analytical methods for only nine as late as 1940. Osborne proved the individuality of proteins within a class. He crystallized 30 plant proteins and showed that they were all different in composition although many were of one class. As early as 1911, together with Harry Wells, he demonstrated the individuality of the proteins by immunological methods. The hemoglobins from different species were shown to be different by Edward Reichert and Amos Brown in 1909 by careful comparison of their respective crystallographic parameters and by Carl Landsteiner and Michael Heidelberger in 1923 by immunological criteria. The interest of the European organic chemists, led by Emil Fischer, lay chiefly in the chemical composition of proteins, which they investigated by examining the products of protein hydrolysis, initially with strong acid or alkali and later by enzymatic cleavage. Before 1935 some 20 amino acids had been identified in protein hydrolysates, but only leucine and tyrosine were not dismissed as artifacts of isolation. Two of the amino acids had been discovered by American scientists because of their essentiality in diets, methionine by John Mueller for the growth of hemolytic streptococcus and threonine by William Rose for the growth of immature rats. One characteristic was held in common by all amino acids; they were all of the L configuration. Consequently there was excitement in 1939 when Fritz Kögl and Christian Erleben reported that in tumor issue they had found D-glutamic acid, as well as the L. I remember a seminar at Cornell Medical College arranged by Dean Burk entitled “Köglism and neo-Köglism” that was attended not only by the protein chemists at Columbia but by those from New Haven, as well, who had attempted to verify this astounding finding. The consensus was that Kögl was wrong. It was my first experience with a probable scientific hoax, presumably perpetrated by a technician although Kögl never withdrew the claim, and it remained controversial. I was one of the co-authors of a paper with Fritz Lipmann casting serious doubt on it by the use of D-amino acid oxidase and deuterium. The peptide bond was generally accepted as a linkage of amino acids in proteins, but whether it was the only linkage was questioned in the period from 1920 to 1940. Fischer, who had invented the term polypeptide and had synthesized many peptides, suggested that there might also be diketopiperazines in native proteins. The chief argument against the universality of the peptide bond was the fact that enzymatic cleavage only worked for some proteins. By 1940, after the specificity of cleavage by proteolytic enzymes had been demonstrated with synthetic peptide substrates by Joseph Fruton and Max Bergmann (another refugee from the Nazis), it was accepted that amino acids in proteins are linked solely by peptide linkage. The whole history of the nature of proteins is full of controversy and strewn with discarded theories, all for the lack of adequate purification and reliable analytical data. Bergmann, a first rate protein chemist, with Leonides Zervas, came to the conclusion in 1938 that there was a periodicity of each amino acid in a protein. This generalization was proven wrong in his own laboratory, when an exception was found by William Stein, and it quietly died. Then Theodor Svedberg, the Swedish inventor of the ultracentrifuge for the determination of molecular weights of proteins, concluded from insufficient data that all proteins were made up of multiple units of molecular weight 17,000, a notion that was discarded as more data contradicting this conclusion became available. The cyclol theory of the three-dimensional structure of globular proteins as a cage-like structure of six-membered rings with the amino acids linked by =N−C(OH)= rather than peptide bonds was espoused with enthusiasm by Dorothy Wrinch, a British topologist, in 1939 and was shot down by Linus Pauling and Carl Niemann. I remember meeting her in New York when I was a postdoctoral fellow. She had invited a group of young biochemists one evening, because she thought they were more open to new ideas and more ready to be persuaded of her unorthodox hypothesis. It is of interest that the three-dimensional structure of proteins was tackled before reliable quantitative data on the amino acid composition was available and considerably before the sequence problem could be undertaken. Because of the absence of methods to study the complexity of proteins, biochemists in the 30s and 40s, well aware that proteins, particularly enzymes, were key molecules in living cells, shied away from them and focused their efforts on small molecules, the substrates and products of enzymatic reactions. The x-ray crystallographers had no such inhibitions. As early as 1926, William Astbury in England, under William Bragg's prodding, subjected many natural fibers, initially keratin of hair, later collagen, silk, and nucleic acid, to x-ray diffraction. The patterns he observed were inadequate, and the methods of analysis available at that time were insufficient to yield structures. Although the structures he proposed proved to be wrong, his enthusiastic espousal of the importance of three-dimensional structures of macromolecules for understanding biological processes persisted. John Kendrew has credited Astbury with being the father of molecular biology. The next development came with the establishment by John Bernal in Cambridge of a group including Dorothy Crowfoot Hodgkin and Max Perutz, and by the mid 1930s they succeeded in obtaining satisfactory x-ray diffraction patterns of pepsin and hemoglobin. Unfortunately, despite a plethora of data, they were unable to analyze them in terms of structure. A different approach was taken in this country by Pauling, who reasoned that in the absence of interpretable x-ray patterns of proteins, the way to go in solving the structural problem was model building. Pauling, a pioneer of structural chemistry, who had elucidated the detailed structure of small inorganic and organic molecules, became interested in proteins in 1935. By 1937, he had published a theoretical paper on the oxygen equilibrium of hemoglobin and its structural interpretation, a paper with Charles Coryell on measurements of the magnetic properties of various forms of hemoglobin, and a paper on the denaturation of proteins with Alfred Mirsky, a protein chemist from Rockefeller Institute. In the latter paper, they stated, “Denaturation is the incomplete or complete unfolding of the polypeptide chains producing molecules that can assume a large number of conformations, giving increased entropy and increased intermolecular interaction [4].” This work initiated his interest in the folding of polypeptide chains. As Pauling relates it, he decided in the summer of 1937, to try to determine the α-keratin structure. From his knowledge of structural chemistry, he would predict the dimensions in a polypeptide chain and then examine the possible conformations until he found one that agreed with the rather fuzzy x-ray data. Although no correct structure of an amino acid or peptide had yet been done, he was convinced that he could reliably predict the dimensions of the peptide group because of the accurately established C−C and C−N bond lengths (known to 0.01 Å) determined in many organic compounds. On the basis of the partial double bond character of the peptide bond, he concluded that it must be planar, which greatly restricts the possible structures. Nevertheless he was unsuccessful in finding a way to fold the polypeptide chain to yield a structure compatible with a 5.1-Å repeat along the fiber axis, which had been deduced from Astbury's α-keratin x-ray diffraction. At this point, he concluded that there must be some structural property of polypeptides that he had not taken into consideration. In the fall of 1937, Robert Corey, who had been working with Ralph Wyckoff at Rockefeller Institute for nine years on x-ray crystallography including proteins, arrived in Pauling's laboratory for a year. He was interested in determining protein structure, and Pauling suggested the task of determining the structure of some simple amino acids and peptides. A photograph of Pauling and Corey at that time, Fig. 2, shows that Pauling was not only ahead of his time in structural chemistry but in dress habits in the laboratory as well. Corey, from the traditional Rockefeller Institute, is wearing a tie and a white dress shirt whereas tieless Pauling seems to be wearing a short-sleeved Hawaiian shirt. With amazing speed, in a little more than a year, Corey, with the aid of two graduate students, succeeded in determining accurate structures of glycine and diketopiperazine. The work continued in Pauling's group into the 1940s until the structures of about a dozen amino acids and some simple peptides had been determined by x-ray diffraction despite slowing because of World War II. A typical structure, that of the amino acid threonine, with exact values of bond lengths and angles, is depicted in Fig. 3, and of the polypeptide chain is shown in Fig. 4. There were no unusual features. In the spring of 1948, Pauling was a visiting professor at Oxford and as he describes it, “I caught cold and was required to stay in bed for about 3 days. After 2 days I had got tired of reading detective stories and science fiction, and I began thinking about the problem of the structure of proteins…. I realized in thinking about the structures of amino acids and peptides determined by Dr. Corey and others that there had been no surprises whatever: every structure conformed to the dimensions,…. bond lengths and bond angles and planarity of the peptide group,…. that I had already formulated in 1937. The N-H-O hydrogen bonds, present in many crystals, were all close to 2.9 Å in length. I thought I would attack the α-keratin problem again. As I lay there in bed I had an idea of a new way of attacking the problem. Back in 1937 I had been so impressed with the fact that the amino acid residues in any position in the polypeptide chain may be any one of 20 different kinds that the idea that with respect to folding they might be nearly equivalent had not occurred to me. I accordingly thought to myself, what would be the consequences of the assumption that all the amino acid residues are structurally equivalent with respect to folding of the polypeptide chain?” Later in his description, he states, “I asked my wife to bring me pencil, paper, and a ruler. By sketching a polypeptide chain on a piece of paper and folding it along parallel lines, I succeeded in finding two structures that satisfied the assumptions. One of these structures was the α-helix, with 4.6 residues per turn, and the other was the γ-helix [5].” That sketch of the polypeptide chain that Pauling made in 1948 is depicted in Fig. 5. You can see his note about folding so that the A part of the structure comes close to the B part of the structure. The repeat along the fiber axis which he then calculated was 5.4 Å per turn, which disagreed with Astbury's value of 5.1. Because of this discrepancy, Pauling delayed publication (the value of 5.4 turned out to be a misinterpretation of the x-ray data). After wrestling unsuccessfully with the discrepancy for almost two years, Pauling and Corey decided to publish their helical structures in 1951 as shown on the lower part of Fig. 5. The α-helix turned out to be one of the significant features of protein structure as evidenced from such segments in the first experimentally determined structures some ten years later of myoglobin by Kendrew and of hemoglobin by Perutz. Pauling has ascribed his success to his adherence to known structural features especially the planarity of the peptide group and to his rejection of the idea, taken over from crystallography, that an acceptable structure should have an integral number of residues per turn. Later he proposed the existence of the parallel β-sheet shown in Fig. 6 and the antiparallel β-sheet shown in Fig. 7, both of which are also found in proteins. His contribution to the structure of proteins continues to make the interpretation of x-ray diffraction structure determinations easier for the practitioners to this day. Furthermore it demonstrated the power of combining model building with experimental data. Pauling's α-helix work also led to a general theory developed by Francis Crick and others of the x-ray pattern expected from a helix, a theory that contributed greatly to solving the structures of nucleic acids, tobacco mosaic virus, and other materials. Let us now consider proteins as enzymes. The view that enzymes and hormones were not proteins was held by many scientists, particularly those who viewed proteins as colloids. Even after the first crystallization of the enzyme urease by James Sumner at Cornell in 1926, an event greeted with skepticism and derision by Richard Willstätter and his followers, and as late 1934, its enzymatic activity was ascribed by them to a small molecular weight impurity. By 1940, 20 enzymes had been crystallized, all proteins, and opposition to acceptance that had been based on the concept that crystallinity was solely an attribute of the inanimate world disappeared. The importance of enzymes in living systems was recognized early in the twentieth century as a result of Eduard Buchner's classical demonstration that fermentation could take place in a cell-free system. I shall not go into the developments in enzymology, a field most active among the European biochemists as they discovered and investigated the enzyme-catalyzed reactions in fermentation, glycolysis, biological oxidations, and the citric acid cycle. Some of these developments have already been alluded to by Howard Gest [1]. I shall limit my presentation to the work of the Coris, who were interested in hormonal regulation of carbohydrate metabolism and whose seminal experiments on glycogen phosphorylase has had such a profound influence on current research on regulation. Fruton has stated, “The Cori laboratory in St. Louis became one of the principal Meccas for aspiring biochemists, and its importance in the institutional development of biochemistry in the United States cannot be exaggerated [6].” In the 40s and 50s, it was undoubtedly the center of enzymology in this country. The Coris became interested in the regulation of carbohydrate metabolism by hormones in the mid 20s when they were working in Buffalo, New York at the State Institute for the Study of Malignant Diseases (now the Roswell Park Memorial Institute). They wanted to know how blood glucose concentration was regulated. They found that insulin increased the conversion of glucose to muscle glycogen but decreased its conversion to liver glycogen. Epinephrine behaved in the opposite manner, decreasing muscle glycogen and increasing liver glycogen. Postulating and later demonstrating that the intermediate formed from muscle glycogen was lactate, the Coris represented the course of events by a cycle that came to be known as the Cori cycle, shown in Fig. 8. This scheme derived from research on the circulation of carbohydrate material in the intact animal was a milestone in the elucidation of carbohydrate metabolism and along with other insights on blood homeostasis, was instrumental in explaining the effect of epinephrine on blood glucose. In later experiments with epinephrine in isolated muscle, they found that there is a concomitant disappearance of inorganic phosphate with the glycogen in muscle preparations, a finding that was confirmed by Parnas in Poland. Upon moving to St. Louis in 1931, the Coris continued research in this area, using extracts of frog muscle. In the first striking result with this system in 1936, they identified 5′adenylic acid (AMP), a compound not directly involved in the reaction, as an obligatory activator of the reaction, foreshadowing the idea that the allosteric activation of enzymes can regulate metabolic processes. Because of a large discrepancy between the phosphorus and reducing power in their careful analyses of the hexose monophosphate product of the reaction, the Coris postulated and later proved (1937) that the compound was glucose-1-phosphate, a hitherto unknown hexose phosphate. The compound became known as the Cori ester. The Coris named their new enzyme, which catalyzed the reaction of glycogen and inorganic phosphate to form glucose-1-phosphate shown in Fig. 9, phosphorylase, in keeping with Parnas's term for the process as phosphorolysis. Their next step was to reverse the reaction with the addition of a small amount of glycogen, producing a large starch-like polysaccharide. They thus established the need of a primer, and more importantly, they had accomplished the first synthesis of a macromolecule in a cell-free system, a sensational contradiction of a long held idea that the biosynthesis of a macromolecule requires energy and consequently can occur only in cells. The conceptual impact was far-reaching, although it is now known that glycogen phosphorylase is on the pathway of glycogen degradation rather than synthesis. Because of their success, subsequent investigators used biochemical approaches to synthesize macromolecules that served them well; Arthur Kornberg mentions it specifically in his description of his search for DNA synthesis [7]. In a brilliant series of papers in 1942–43, the Coris, in collaboration with Arda Green, crystallized phosphorylase and, among other aspects, characterized its two forms, a and b, and their role in metabolic regulation. They found that the b form was active only with AMP and that the conversion of b to a was enzyme-catalyzed. Furthermore the Coris postulated that interconversion plays a significant role in metabolic regulation, because the inactive form is found in resting muscle, and the active form is found in contracting muscle. Subsequent work involved the effect of hormones on the interconversion of the two forms. In the mid-fifties, two of the Coris' students who had worked on phosphorylase in the Cori lab, discovered that the conversion of b to a involved phosphorylation of the enzyme. These students were Earl Sutherland, working with the liver enzyme and Edwin Krebs, in collaboration with Edmond Fischer, working on the muscle enzyme. This was the first example of metabolic regulation by protein phosphorylation, the most prevalent mechanism of regulation known today. Kreb's work led to the elucidation of the complex cascade of regulating enzymes involved in the conversion of b to a, and Sutherland's work led to the discovery of cyclic AMP and his concept of the second messenger, which illuminates the area of signal transduction. Thus the Coris early work in the 1920s on the regulation of carbohydrate metabolism by epinephrine has had enormous consequences on current biochemical research. Unfortunately, in the limited time available, I cannot cover many important areas of research of the first half of the 20th century. However, I would be remiss if I did not mention three papers published in the 1940s during World War II with far-reaching consequences, introducing new concepts and opening up new areas of research for the post-war period. They are as follows: F. Lipmann (1941), Metabolic generation and utilization of phosphate bond energy [8]; G.W. Beadle, E. L. Tatum (1941), Genetic control of biochemical reactions in Neurospora [9], and last but not least, O. T. Avery, C. M. MacLeod, M. McCarty (1944), Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a deoxyribonucleic acid fraction isolated from pneumococcus Type III [10]. Lipmann's paper not only introduced the concept of “high energy phosphate bonds” but predicted that the biosynthesis of proteins and other macromolecules would require energy supplied by the cleavage of a “high energy bond” of nucleoside triphosphates. His prediction was correct. The Beadle and Tatum paper introduced a new approach to biochemical genetics; instead of starting with inheritable traits and trying to find the biochemical basis, they reversed the process by starting with a biochemical reaction in a suitable organism, Neurospora crassa, and identifying the gene responsible. Finally the Avery, Macleod, McCarty paper indicated that not protein, but nucleic acid, until now considered an insignificant player in living systems, was the carrier of genetic information. The limiting factor for research progress until after World War II was the unavailability of materials, of quantitative methods for separation and purification, and of instrumentation. Although 14C was discovered in 1940, it did not become generally available until after the war when it could be produced cheaply, in quantity, in atomic reactors. Sophisticated counters for measuring radioactivity were marketed commercially shortly after the war, as well, making 14C a common tool of research investigations in most biochemical laboratories. I have already mentioned that the lack of methods for quantitatively analyzing the hydrolytic products of proteins led to incorrect speculation of protein structure even by outstanding scientists. One of the greatest boons to biochemists has been the development of chromatographic methods for the separation and purification of metabolites and macromolecules. The seminal paper describing partition chromatography was published by the British scientists, Archer Martin and Richard Synge, in 1941 [11]. It was in 1937 that Arne Tiselius in Sweden devised the method of moving boundary electrophoresis for separating proteins differing in electric charge [12]. Currently, gel electrophoresis has supplanted the Tiselius method to become one of the most powerful and generally used analytical procedures for proteins and nucleic acids. It was Warburg, one of the great German enzymologists of the first half of the twentieth century, who developed an ultraviolet spectrophotometer for enzyme assays that replaced his manometric methods. However, it was such a complicated instrument that its use was confined to his own laboratory, and it was not until 1942, when the Beckmann DU (ultraviolet) spectrophotometer became commercially available, that spectrophotometers became the universal work horse of enzymologists. The mass spectrometer was first introduced in biochemical research in 1937 by Rittenberg who constructed one for tracer studies with stable isotopes. It was a primitive instrument of limited usefulness compared with the current explosion in applications of modern mass spectrometry. Even the computer, which impinges on almost every research activity in biochemistry today, was first introduced in a primitive form for simulation of enzyme kinetics by Britton Chance in 1943 [13]. There are many other methods and techniques that were first introduced in the period under discussion but because of the constraints of time, I have highlighted only a few of the most spectacular ones. In closing, I hope that I have convinced you that some of the most active and exciting areas of research in biochemistry after 1950 had their origins in the concepts and experimental methods developed in the 1930s and 1940s. The rates of synthesis and degradation of fatty acids. R. Schoenheimer, D. Rittenberg (1936) J. Biol. Chem. 114, 381–396. Photograph of Linus Pauling and Robert Corey in 1937. L. Pauling (1996) Chem. Intell. 2, 32–38. The structure of threonine. D. P. Shoemaker, J. Donohue, V. Shoemaker, R. B. Corey (1950) J. Am. Chem. Soc. 72, 2328–2348. The structure of the polypeptide chain. L. Pauling, R. B. Corey, H. R. Branson (1951) Proc. Natl. Acad. Sci. U. S. A. 37, 205–211. Pauling's 1948 sketch of the polypeptide chain and the 1951 published version of the α-helix [5]. The structure of the parallel β-sheet. L. Pauling, R. B. Corey (1951) Proc. Natl. Acad. Sci. U. S. A. 37, 729–740. The structure of the antiparallel β-sheet. L. Pauling, R. B. Corey (1951) Proc. Natl. Acad. Sci. U. S. A. 37,738. The Cori cycle (1928). The glycogen phosphorylase reaction.