Protein Science (1992), I , 1526-1530. Cambridge University Press. Printed in the USA. Copyright 0 1992 The Protein Society
RECOLLECTIONS
Memories of early days in protein science, 1926-1940
JOHN T. EDSALL Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 (RECEIVED July 6,1992; ACCEPTED July 15, 1992)
I got my start in protein chemistry asa third-year medical studentat Harvard,in the early fall of 1926. Early that summer I had returned to HarvardMedical School after 2 years in Cambridge, England, where I had worked in the great laboratory headedby Sir Frederick Hopkins. I had taken the Part I1 course inbiochemistry and had done a bit of research on phosphates in muscle-a rather trifling affair, thoughit taught me much.For a future protein physical chemist, who did not yet know which way he was headed, my most important contact was surely G.S. Adair, a very shy butfriendly and highly gifted young man, 6 years olderthan myself. He was in the process of settling, by osmotic pressure measurements, a problem that had caused enormous confusion:what was the true molecular weight of hemoglobin? From the iron content of the protein, about 0.33%, one could figure that the minimum value was roughly 16,000; but it could be any integral multiple of that number. Most people then believed, on inadequate grounds, that the true value was indeed near 16,000, with just one oxygen-binding heme site per molecule. In his laboratory, Adair had to work several years before he could consistently make semipermeable membranes that did not leak; there were of course other problems also. On the theoretical side hewas probably the only biochemist of that time who had really read the great thermodynamic treatiseof J. Willard Gibbs; and he was certainly the first person to realize that Gibbs had formulated the Donnan equilibrium,35 years before Donnan. Knowledge of the Gibbs-Donnan effect was certainly vital for getting the true molecular weight of a protein, with its numerous electric charges. After about 5 years, Adair felt sure of his results and reported that thehemoglobin molecule was four times as big as people had generally thought. Manysenior people were startled. Some months later TheSvedberg in Uppsala, who had just started to apply his ultracentrifuge to Reprint requests to: John T. Edsall, Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138.
the studyof proteins, reported the samemolecular weight; he did not yet know of Adair’s work. Adair went on to formulate his equations for ligand binding by hemoglobin, recognizing the positive (cooperative) interactions between the four successive binding constants. Neither he nor anyone else, at that time, however, could propose a mechanism to explain the nature of the interactions involved. Adair’s equations remain fundamental today.
Starting on protein research
Coming back to Harvard for the third year of medical school- I had donemost of the second-year studies while in England-I was eager to do a bit of research in what spare time I had in that clinical year. I had notyet focused on a choice of problem, but I had developed an interest in the workings of muscle. I went to talk with the physiologist Alfred c. Redfield, under whose guidance I had done a little piece of research on themuscular activity of the heartmuscles of tortoises, asa first-year medical student. Thatwas what had fired my interest in muscle in the first place. Redfield remarked “I think the most neglected aspect of researchon muscle today is the studyof muscle proteins. Edwin Cohn,in the labclose by, has had a student workingon aninteresting protein from muscle; but the student hadto leave, to dofull time clinical work. (The student was Bill Salter, who later became Professor of Pharmacology atYale.) Ifyou are interested, why not talk to Cohn, and see if he would like to take you on?” That conversation with Redfield was a turning point in my life. I did see Cohn, andhe accepted me (Fig. 1). The study of thatmuscle protein, in thelimited time I had to work on it, became quite fascinating. Its solutions were highly viscous, even when pretty dilute, and it never showed any tendency to formcrystals. Its solubility was extremely sensitive to the ionic strength. In thenext year, however, when I had much more time for research, the situationbecame exciting, when a young man of extraordinary talents, Alex von Muralt, arrived
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Protein science, 1926-1940
1527
Fig. 1. John T. Edsall at work in the Department of Physical Chemistry, Harvard Medical School. Photograph taken by E.
Kendall Bragg in May 193 I .
from Switzerland to work in Cohn’s laboratory. He had Henderson, Cohn, and the Department taken a Ph.D. in physics, before going into physiology, of Physical Chemistry and was a master of optical techniques, and much else, including instrumentdesign. He was working on double The Department of Physical Chemistry at HarvardMedrefraction of muscle; one daywe decided to have a look ical School had been established about 1920, with Lawat my protein solutions for possible double refraction. rence J. Henderson (1878-1942) as its head. Henderson There was none in the solution atrest, but gentle stirring was an extraordinary man - biochemist, physiologist, with a rod evoked it strongly, along the streamlines pro- teacher of the history of science, philosopher of science duced by the stirring. The meaning was clear; the solu(in his books on The Fitness of the Environment and tion contained large asymmetric particles oriented, moreThe Order of Nature), and in his last years a sociologist, or less in parallel, by the velocity gradients in the liquid. deeply influenced by the work of Vilfredo Pareto. For me When the stirring stopped, thebirefringence disappeared his greatest achievement was his work on blood and its gradually, in a matter of seconds, as the oriented partirespiratory function, as a physicochemical system of incles became disoriented by Brownian motion. That was teracting components,in which the activity of each comthe fundamental fact,but it took us 2 years to define what ponent is a function of all the others; a modification in was going on in quantitative terms and explore its ramifione induces changes in all. He and his collaborators cations. Then we wrote up ourresults in three papers that worked out all these relations in detail, in quantitative terms. His vision of a biological system-inevitably, of appeared in the Journal of Biological Chemistry. We had established a relation between the birefringent elements course, simplified even as a model of blood, and focused of the muscle fiber andthe asymmetric particles,derived on the transportof oxygen and COz-is, I believe, a prefrom themuscle, in our protein solution. Thiswas a start cursor of other biological systems analyses, of far greater for studies by others that carried the study of the struccomplexity, that will be done in the future. Henderson was for me a great teacher; I had taken his biochemistry course ture and function of muscle vastly further. Happily for as an undergraduate,which provided great mental stimme it was also the startof a lifelong friendship with Alex von Muralt. ulus, and I got much helpful guidance from him later.
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Henderson’s involvement withblood as a system, however, drew him away from the Department of Physical Chemistry, first to Massachusetts General Hospital, with Dr. Arlie Bock and others; later (to the surprise of many of us) to the Fatigue Laboratory of the HarvardBusiness School, where David Bruce Dill presided over the big laboratory in the basement, and Henderson had an office on the floor above. The Rockefeller Foundation put up the money for all this and supported theFatigue Laboratory until its dissolution in 1946, 4 years after Henderson’s death. To run the laboratory in the Department of Physical Chemistry at the Medical School Henderson had chosen a young man, Edwin J. Cohn (1892-1953), who had been a graduate student with him. Cohn had made up his mind, at anearly stage, to dedicate his scientific life to the study of proteins, and Henderson supported him inthe decision, which was most unusual in those days. Biochemists whose work really centered on proteins were rare birds. Most chemists, in a somewhat tentative fashion, believed inthe view of Emil Fischerand of Franz Hofmeister, that amino acid residues in proteins were held together by peptide linkages, and the fact that proteolytic enzymes also split peptide linkages in small synthetic peptides, as shown by Bergmann and Fruton, strengthened confidence in the idea. There were rival hypotheses, most notably the ingenious “cyclol structures” of Dorothy Wrinch, which caused much excitementfor awhile but eventually proved to be irrelevant to proteins. Cohn, having completed his Ph.D., had gone to work in the laboratory of Thomas B. Osborne, Director of the Connecticut Agricultural Station in New Haven, the foremost protein chemist inthe country at thattime. The First World Warinterrupted that, and he returned to Harvard to work under Henderson in a war-related project for making bread from a wide variety of grains. After the war, Cohn got a National Research Council Fellowship and went to Denmark to work with the great protein chemist S.P.L. S~rrensen.Returning to Harvard, Cohn launched studies on protein solubility, particularly the striking influences of added salts and changes of pH. He was greatly influenced, and oftenguided, by his close friend George Scatchard of MIT, who had a profound knowledge of the physical chemistry of solutions, andof the Debye-Huckel theory of interionic attractions. Cohn also studied titration curves of proteins, correlating the pH regions of strong proton binding with the (then imperfectly known) amino acid composition of the protein. Henderson, as he becamedrawn into his workon blood elsewhere, had made plain his confidence in Cohn, who soon became the de facto head of the department. Before long he became a full professor and head of the department. Cohn was ambitious and aggressive, and he made enemies in several quarters. He was gifted in his ability to spot a significant area of research and bring together a group of investigators of diverse and complementarytalents, whose interplay led to important achievements. He
J. T. Edsall
did this in the 1930s, as I shall briefly describe in the closing section of this article; and he did it again, on amuch larger scale, in the war years, in the blood plasma fractionation program that he initiated and led. Under the immense pressures of the wartime project he could often infuriate the younger members of the group, as he criticized them and urged them on, and then again, within a matter of minutes, he could become relaxed and genial. In that complex enterprisehe generally showed goodjudgment in knowing who was the right person to doa given job. I have written more about him elsewhere. Were proteins really molecules? It may seemhard to believe, for the young protein chemists of today, that therewas a school of thought, among the colloid chemists,which heldthat proteins were not true molecules, but simply heterogeneous aggregates of various small molecules, presumably peptides of moderate size. Even the organic chemists found it almost incredible that true macromolecules could exist; the great Emil Fischef tended to believe that there was an inherent limit to the length of a stable peptide chain, which he took to be around 30-40 residues. There was enormous resistance to Hermann Staudinger, among most organic chemists in the early 192Os, when he claimed that the synthetic polymers that he had prepared were true macromolecules, containing hundreds of repeating units held together by covalent bonds. The resistance soon gave way.Within another decade the correctness of Staudinger’s views was generally acknowledged. In fact,people likeHenderson and Cohn never, I think, took the views of the colloidalists seriously. Osborne, of the older generation, had the same attitude, from what Cohn told me. Osborne had done extensive workon plant seed globulins and had crystallized many of them. People whohad worked with crystals were unlikelyto believe that proteins were merely miscellaneous aggregates of small molecules. The most eminent member of the colloidal school was The Svedberg, in Uppsala, and he became converted to the molecular point of view as soon as he started studying proteins in his ultracentrifuge. I remember well the day, about 1930, when Svedberg came for a visit to our laboratory and told us of an argument he and Cohn had had, about 10 years before, when Cohn was still a young postdoc working with Ssrensen. Svedberg was then still developing the ultracentrifuge, eager to use it on proteins when it would be ready, a few years later. An argument developed. Svedberg expected to see protein solutions sedimenting with broad diffuse boundaries, corresponding to many different molecular weights. Cohn challenged him on this and predicted that he wouldfind well-defined boundaries in good protein preparations, indicating that the molecules were all of one size. Svedberg said to us: “Of course I soon found that Cohnwas right,” andhe became one of the most powerful advocates of the view that
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Protein science, 1926- 1940 proteins were genuine molecules with well-defined, specific structures. Search for understanding of structures and forces, 1930-1940
About 1930, our research in the Department of Physical Chemistry took anew turn. Studies of proteins continued actively, but prime concern shifted for some years to the smaller molecules, the aminoacids and peptides, and other closely related substances. They contained electric charges, polar groups with no net charge, andvarious nonpolar side chains. The surrounding medium, for most proteins in their natural state,was largely water, with a sprinkling of inorganic ions at concentrations usually on the order of 0.1 M or less. How did these molecules interactwith water and with the ions dissolved in it? Thesolubility of proteins, we knew, was often profoundly influenced by added salt. Did amino acids and peptides show similar, but probably smaller, effects? How did the nonpolar side chains of amino acids such as leucine affect their interactions with water and othersubstances? These questions indicate some of the problems we investigated, but at the time we did not know enough to ask such coherent questions. We had to learn what questions to ask, alongwith the search for the answers. The group thatgot involved in all this grew over several years (Fig. 2). Jeffries Wyman, my closest friend, had returned to the HarvardBiology Department after getting his Ph.D. in London with A.V. Hill. He had then worked for a year with us in Cohn's laboratory while teaching in Cambridge. T.L. McMeekin, who arrivedsoon after,was to make a series of important contributions. George
Scatchard was, as ever, deeply involved with our problems, and a young man, then also at MIT, JohnG . (Jack) Kirkwood, was to play a major role as a theorist. Jesse P. Greenstein, who arrived in our laboratory around 1930, proved highly skilled in peptide synthesis and was a versatile member of the group. (Later he moved to the National Cancer Institute and became the leader of its chemistry division.) Afew years later LarryOncley came over to us from MIT (he had completed his Ph.D. with J.W. Williams in Wisconsin) and soon became a major contributor to the studyof dielectric constants of proteins and their relaxation times, which gave important information concerning the shapes of the protein molecules. He later played a key role in the war years, particularly in his work on gamma-globulins and plasma lipoproteins. Still later, John D. Ferry arrived,working at first with Oncley. He alsowas to play a major role in the plasma program, particularly in the development of fibrin foam and fibrin film, which proved of great value to neurosurgeons. A gifted graduate student from the Harvardbiology department, Peter Morrison, worked on those projects too and contributed much to them. I also was closelyinvolved in this work. Much of our interest in the years around 1930 arose from two important papers on the ionization of amino acids, one by E.Q. A d a m in 1916, and the other, more comprehensive,paper by Niels Bjerrum 7 years later. Before that time it was generally assumed that a simple isoelectric amino acid could be describedby the formula H,N-CHR-COOH. Adams and Bjerrum both gave powerful reasons for believing that the true formula was +H,N-CHR-COO-. Such a structure should be a dipole
Y
Lu
L
\ d
Fig. 2. Harvard Medical School, Departments of Physiology and Physical Chemistry, 1934. Members of the Department of Physical Chemistry are: front row: Ronald M. Ferry (second from left), E.J. Cohn (fourth), J.T. Edsall (second from right). with Walter Cannon, headof the Departmentof Physiology, is fifth from left. Second row: J.P. Greenstein (extreme right) T.L. McMeekin, next to him. Fourth row: department secretary AnnaB. Curtiss (second from right). Lastrow: technicians George Greco, John H. Weare, and Muriel H. Blanchard (three, four, and five from left).
1530 of very high electric moment, with positive and negative ionic charges, separated by about 3 A. A series of peptides, as the number of residues increased, would haveincreasing separation of the charges with each added residue; so they could have very highdipole moments indeed. We called these molecules dipolar ions; I now think that ionic dipole would be a better term and will use it here. Jeff Wyman undertook to measure the dielectric constants of amino acids and peptides in water solution, to test thishypothesis. This required a new approach; the existing methods could not handle solutions of such relatively highconductivity as these solutions. Within 2 years he had solved the problem and studied a series of amino acids and peptides with McMeekin. The results spectacularly supported theionic dipole hypothesis; the dielectric constants of these solutions were linear functions of the concentration. A molar solution of an a-aminoacid gave a value 22.5 units higher than that of water; for dipeptides the corresponding value was 70, for tripeptides 113, and so on. Wyman’s interpretation of his data stimulated Lars Onsager to produce the first good theory of the dielectric constants of polar liquids. I took adifferent approach to the ionic dipole hypothesis, by the study of Raman spectra of aqueous solutions of amino acids and related compounds. The characteristic vibrational frequencies of the amino and carboxyl groups in the charged and uncharged states were easy to obtain, from amines and carboxylic acids, and the comparison with isoelectricamino acids immediately showed that both groups were charged. Raman spectra were also valuable for studying many other problems, such as the effects of exchanging deuterium for hydrogen; but these need not be considered here. There were other implications of the ionic dipolar structure ofthe amino acids and peptides. The apparent molar volumes ofa-amino acids in water proved to be some 13 cm3smaller than those of isomeric polar but uncharged compounds; a comparison of glycine and glycolamide was the simplest example. The difference was due to the electrostriction of water moleculesthat were squeezedtightly around the charged groups by the electric fieldof the iondipole. The same mechanism also produced a marked lowering of the heat capacity of the system. These effects, of course, were already well known for ions. McMeekin, with various other members of the laboratory, carried out along seriesof solubility studiesof amino acids in various media, varyingboth the ionic strength and the dielectric constant of the solvent medium; the latter was variedby adding alcohol or acetone. Jack Kirkwood, sometimes in association with Scatchard, developed for dipoles an extension of the Debye-Huckel theory of interionic attractions and studied several modelsfor dipoles, at least one of which gavea very good fit to McMeekin’s data. Kirkwood’s brilliant theoretical analysis was one of the first steps in hisoutstanding career, culminating in the chemistry department at Yale, and ending tragically in his death from cancer at the age of 52.
J. T. Edsall
McMeekin also studied the relative solubility of amino acids in water and organic solvents of lower dielectricconstant, such as ethanol. Nonpolar side chains shifted the relative solubility in water and ethanol according to a simple rule; glycine was some 2,000 times as soluble in water as in ethanol; for alanine the ratio was near 700; for aaminobutyric acid it was near 250. Each added CH2 group altered the ratio by a factor close to three. This was another example of the rule formulated by Isidor Traube in 1891, as a result of his studies of the lowering of the surface tension of waterby members of a homologous series, suchas alcohols or fatty acids, containing a polar end group and a hydrocarbon chain. The concentration in the solution that was required to lower the surface tension by a given amount decreased by a factor close to three for each added CH2 group in the chain. Traube’s work was perhaps the first description of whatwe now knowas hydrophobic interactions. I have written about his life and work elsewhere. I published a small contribution to the hydrophobic effect in 1935. Lookingat other people’s data for molar heat capacities in some homologous series of pure organic liquids, and then at the data for the apparent molar heat capacities of the same compounds in aqueous solution, it was clear that the increment in molar C, per CH2 group (in cal mol”) was far larger in aqueous solution (20-30) than in the organic liquid (5-8). Apparently none of the workers involved had commented on this striking difference. I did not know what itmeant, but the difference was startling, and I called attention to it in a two-page paper in the Journal of the American Chemical Society. In recent years such people as Julian Sturtevant, Ingemar Wadso, Stanley Gill, and Peter Privalov have vastly extended the data and have shown that these striking increases in heat capacity, on exposure of hydrophobic groups to water, as for instance in protein unfolding, represent a general phenomenon. There were ofcourse other studies on what we now call hydrophobic effects in the 1930s, such as the important work of J.A.V. Butler on the enthalpy and entropy changes for aseries ofalcohols dissolved inwater. However our thoughtson these matters were for themost part fragmentary. The first clear and coherent look at these phenomena, in their relationto protein structure and function, did not come until 1959, in Walter Kauzmann’sgreat paper in Vol. 14 of Advances in Protein Chemistry. With this backward look at some of the early days in protein chemistry I conclude these unsystematic reminiscences. With the coming of war in Europe in 1939, and the entry of the United States into the war in 1941, we entered a different era in which the Department of Physical Chemistry was mobilized for scientific service inthe war, like thousands of other laboratories. Much has been written about the blood plasmafractionation project and what it accomplished, in war and later in peace, but thatis another story.
RECOLLECTIONS
Memories of early days in protein science, 1926-1940
JOHN T. EDSALL Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 (RECEIVED July 6,1992; ACCEPTED July 15, 1992)
I got my start in protein chemistry asa third-year medical studentat Harvard,in the early fall of 1926. Early that summer I had returned to HarvardMedical School after 2 years in Cambridge, England, where I had worked in the great laboratory headedby Sir Frederick Hopkins. I had taken the Part I1 course inbiochemistry and had done a bit of research on phosphates in muscle-a rather trifling affair, thoughit taught me much.For a future protein physical chemist, who did not yet know which way he was headed, my most important contact was surely G.S. Adair, a very shy butfriendly and highly gifted young man, 6 years olderthan myself. He was in the process of settling, by osmotic pressure measurements, a problem that had caused enormous confusion:what was the true molecular weight of hemoglobin? From the iron content of the protein, about 0.33%, one could figure that the minimum value was roughly 16,000; but it could be any integral multiple of that number. Most people then believed, on inadequate grounds, that the true value was indeed near 16,000, with just one oxygen-binding heme site per molecule. In his laboratory, Adair had to work several years before he could consistently make semipermeable membranes that did not leak; there were of course other problems also. On the theoretical side hewas probably the only biochemist of that time who had really read the great thermodynamic treatiseof J. Willard Gibbs; and he was certainly the first person to realize that Gibbs had formulated the Donnan equilibrium,35 years before Donnan. Knowledge of the Gibbs-Donnan effect was certainly vital for getting the true molecular weight of a protein, with its numerous electric charges. After about 5 years, Adair felt sure of his results and reported that thehemoglobin molecule was four times as big as people had generally thought. Manysenior people were startled. Some months later TheSvedberg in Uppsala, who had just started to apply his ultracentrifuge to Reprint requests to: John T. Edsall, Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138.
the studyof proteins, reported the samemolecular weight; he did not yet know of Adair’s work. Adair went on to formulate his equations for ligand binding by hemoglobin, recognizing the positive (cooperative) interactions between the four successive binding constants. Neither he nor anyone else, at that time, however, could propose a mechanism to explain the nature of the interactions involved. Adair’s equations remain fundamental today.
Starting on protein research
Coming back to Harvard for the third year of medical school- I had donemost of the second-year studies while in England-I was eager to do a bit of research in what spare time I had in that clinical year. I had notyet focused on a choice of problem, but I had developed an interest in the workings of muscle. I went to talk with the physiologist Alfred c. Redfield, under whose guidance I had done a little piece of research on themuscular activity of the heartmuscles of tortoises, asa first-year medical student. Thatwas what had fired my interest in muscle in the first place. Redfield remarked “I think the most neglected aspect of researchon muscle today is the studyof muscle proteins. Edwin Cohn,in the labclose by, has had a student workingon aninteresting protein from muscle; but the student hadto leave, to dofull time clinical work. (The student was Bill Salter, who later became Professor of Pharmacology atYale.) Ifyou are interested, why not talk to Cohn, and see if he would like to take you on?” That conversation with Redfield was a turning point in my life. I did see Cohn, andhe accepted me (Fig. 1). The study of thatmuscle protein, in thelimited time I had to work on it, became quite fascinating. Its solutions were highly viscous, even when pretty dilute, and it never showed any tendency to formcrystals. Its solubility was extremely sensitive to the ionic strength. In thenext year, however, when I had much more time for research, the situationbecame exciting, when a young man of extraordinary talents, Alex von Muralt, arrived
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Protein science, 1926-1940
1527
Fig. 1. John T. Edsall at work in the Department of Physical Chemistry, Harvard Medical School. Photograph taken by E.
Kendall Bragg in May 193 I .
from Switzerland to work in Cohn’s laboratory. He had Henderson, Cohn, and the Department taken a Ph.D. in physics, before going into physiology, of Physical Chemistry and was a master of optical techniques, and much else, including instrumentdesign. He was working on double The Department of Physical Chemistry at HarvardMedrefraction of muscle; one daywe decided to have a look ical School had been established about 1920, with Lawat my protein solutions for possible double refraction. rence J. Henderson (1878-1942) as its head. Henderson There was none in the solution atrest, but gentle stirring was an extraordinary man - biochemist, physiologist, with a rod evoked it strongly, along the streamlines pro- teacher of the history of science, philosopher of science duced by the stirring. The meaning was clear; the solu(in his books on The Fitness of the Environment and tion contained large asymmetric particles oriented, moreThe Order of Nature), and in his last years a sociologist, or less in parallel, by the velocity gradients in the liquid. deeply influenced by the work of Vilfredo Pareto. For me When the stirring stopped, thebirefringence disappeared his greatest achievement was his work on blood and its gradually, in a matter of seconds, as the oriented partirespiratory function, as a physicochemical system of incles became disoriented by Brownian motion. That was teracting components,in which the activity of each comthe fundamental fact,but it took us 2 years to define what ponent is a function of all the others; a modification in was going on in quantitative terms and explore its ramifione induces changes in all. He and his collaborators cations. Then we wrote up ourresults in three papers that worked out all these relations in detail, in quantitative terms. His vision of a biological system-inevitably, of appeared in the Journal of Biological Chemistry. We had established a relation between the birefringent elements course, simplified even as a model of blood, and focused of the muscle fiber andthe asymmetric particles,derived on the transportof oxygen and COz-is, I believe, a prefrom themuscle, in our protein solution. Thiswas a start cursor of other biological systems analyses, of far greater for studies by others that carried the study of the struccomplexity, that will be done in the future. Henderson was for me a great teacher; I had taken his biochemistry course ture and function of muscle vastly further. Happily for as an undergraduate,which provided great mental stimme it was also the startof a lifelong friendship with Alex von Muralt. ulus, and I got much helpful guidance from him later.
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Henderson’s involvement withblood as a system, however, drew him away from the Department of Physical Chemistry, first to Massachusetts General Hospital, with Dr. Arlie Bock and others; later (to the surprise of many of us) to the Fatigue Laboratory of the HarvardBusiness School, where David Bruce Dill presided over the big laboratory in the basement, and Henderson had an office on the floor above. The Rockefeller Foundation put up the money for all this and supported theFatigue Laboratory until its dissolution in 1946, 4 years after Henderson’s death. To run the laboratory in the Department of Physical Chemistry at the Medical School Henderson had chosen a young man, Edwin J. Cohn (1892-1953), who had been a graduate student with him. Cohn had made up his mind, at anearly stage, to dedicate his scientific life to the study of proteins, and Henderson supported him inthe decision, which was most unusual in those days. Biochemists whose work really centered on proteins were rare birds. Most chemists, in a somewhat tentative fashion, believed inthe view of Emil Fischerand of Franz Hofmeister, that amino acid residues in proteins were held together by peptide linkages, and the fact that proteolytic enzymes also split peptide linkages in small synthetic peptides, as shown by Bergmann and Fruton, strengthened confidence in the idea. There were rival hypotheses, most notably the ingenious “cyclol structures” of Dorothy Wrinch, which caused much excitementfor awhile but eventually proved to be irrelevant to proteins. Cohn, having completed his Ph.D., had gone to work in the laboratory of Thomas B. Osborne, Director of the Connecticut Agricultural Station in New Haven, the foremost protein chemist inthe country at thattime. The First World Warinterrupted that, and he returned to Harvard to work under Henderson in a war-related project for making bread from a wide variety of grains. After the war, Cohn got a National Research Council Fellowship and went to Denmark to work with the great protein chemist S.P.L. S~rrensen.Returning to Harvard, Cohn launched studies on protein solubility, particularly the striking influences of added salts and changes of pH. He was greatly influenced, and oftenguided, by his close friend George Scatchard of MIT, who had a profound knowledge of the physical chemistry of solutions, andof the Debye-Huckel theory of interionic attractions. Cohn also studied titration curves of proteins, correlating the pH regions of strong proton binding with the (then imperfectly known) amino acid composition of the protein. Henderson, as he becamedrawn into his workon blood elsewhere, had made plain his confidence in Cohn, who soon became the de facto head of the department. Before long he became a full professor and head of the department. Cohn was ambitious and aggressive, and he made enemies in several quarters. He was gifted in his ability to spot a significant area of research and bring together a group of investigators of diverse and complementarytalents, whose interplay led to important achievements. He
J. T. Edsall
did this in the 1930s, as I shall briefly describe in the closing section of this article; and he did it again, on amuch larger scale, in the war years, in the blood plasma fractionation program that he initiated and led. Under the immense pressures of the wartime project he could often infuriate the younger members of the group, as he criticized them and urged them on, and then again, within a matter of minutes, he could become relaxed and genial. In that complex enterprisehe generally showed goodjudgment in knowing who was the right person to doa given job. I have written more about him elsewhere. Were proteins really molecules? It may seemhard to believe, for the young protein chemists of today, that therewas a school of thought, among the colloid chemists,which heldthat proteins were not true molecules, but simply heterogeneous aggregates of various small molecules, presumably peptides of moderate size. Even the organic chemists found it almost incredible that true macromolecules could exist; the great Emil Fischef tended to believe that there was an inherent limit to the length of a stable peptide chain, which he took to be around 30-40 residues. There was enormous resistance to Hermann Staudinger, among most organic chemists in the early 192Os, when he claimed that the synthetic polymers that he had prepared were true macromolecules, containing hundreds of repeating units held together by covalent bonds. The resistance soon gave way.Within another decade the correctness of Staudinger’s views was generally acknowledged. In fact,people likeHenderson and Cohn never, I think, took the views of the colloidalists seriously. Osborne, of the older generation, had the same attitude, from what Cohn told me. Osborne had done extensive workon plant seed globulins and had crystallized many of them. People whohad worked with crystals were unlikelyto believe that proteins were merely miscellaneous aggregates of small molecules. The most eminent member of the colloidal school was The Svedberg, in Uppsala, and he became converted to the molecular point of view as soon as he started studying proteins in his ultracentrifuge. I remember well the day, about 1930, when Svedberg came for a visit to our laboratory and told us of an argument he and Cohn had had, about 10 years before, when Cohn was still a young postdoc working with Ssrensen. Svedberg was then still developing the ultracentrifuge, eager to use it on proteins when it would be ready, a few years later. An argument developed. Svedberg expected to see protein solutions sedimenting with broad diffuse boundaries, corresponding to many different molecular weights. Cohn challenged him on this and predicted that he wouldfind well-defined boundaries in good protein preparations, indicating that the molecules were all of one size. Svedberg said to us: “Of course I soon found that Cohnwas right,” andhe became one of the most powerful advocates of the view that
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Protein science, 1926- 1940 proteins were genuine molecules with well-defined, specific structures. Search for understanding of structures and forces, 1930-1940
About 1930, our research in the Department of Physical Chemistry took anew turn. Studies of proteins continued actively, but prime concern shifted for some years to the smaller molecules, the aminoacids and peptides, and other closely related substances. They contained electric charges, polar groups with no net charge, andvarious nonpolar side chains. The surrounding medium, for most proteins in their natural state,was largely water, with a sprinkling of inorganic ions at concentrations usually on the order of 0.1 M or less. How did these molecules interactwith water and with the ions dissolved in it? Thesolubility of proteins, we knew, was often profoundly influenced by added salt. Did amino acids and peptides show similar, but probably smaller, effects? How did the nonpolar side chains of amino acids such as leucine affect their interactions with water and othersubstances? These questions indicate some of the problems we investigated, but at the time we did not know enough to ask such coherent questions. We had to learn what questions to ask, alongwith the search for the answers. The group thatgot involved in all this grew over several years (Fig. 2). Jeffries Wyman, my closest friend, had returned to the HarvardBiology Department after getting his Ph.D. in London with A.V. Hill. He had then worked for a year with us in Cohn's laboratory while teaching in Cambridge. T.L. McMeekin, who arrivedsoon after,was to make a series of important contributions. George
Scatchard was, as ever, deeply involved with our problems, and a young man, then also at MIT, JohnG . (Jack) Kirkwood, was to play a major role as a theorist. Jesse P. Greenstein, who arrived in our laboratory around 1930, proved highly skilled in peptide synthesis and was a versatile member of the group. (Later he moved to the National Cancer Institute and became the leader of its chemistry division.) Afew years later LarryOncley came over to us from MIT (he had completed his Ph.D. with J.W. Williams in Wisconsin) and soon became a major contributor to the studyof dielectric constants of proteins and their relaxation times, which gave important information concerning the shapes of the protein molecules. He later played a key role in the war years, particularly in his work on gamma-globulins and plasma lipoproteins. Still later, John D. Ferry arrived,working at first with Oncley. He alsowas to play a major role in the plasma program, particularly in the development of fibrin foam and fibrin film, which proved of great value to neurosurgeons. A gifted graduate student from the Harvardbiology department, Peter Morrison, worked on those projects too and contributed much to them. I also was closelyinvolved in this work. Much of our interest in the years around 1930 arose from two important papers on the ionization of amino acids, one by E.Q. A d a m in 1916, and the other, more comprehensive,paper by Niels Bjerrum 7 years later. Before that time it was generally assumed that a simple isoelectric amino acid could be describedby the formula H,N-CHR-COOH. Adams and Bjerrum both gave powerful reasons for believing that the true formula was +H,N-CHR-COO-. Such a structure should be a dipole
Y
Lu
L
\ d
Fig. 2. Harvard Medical School, Departments of Physiology and Physical Chemistry, 1934. Members of the Department of Physical Chemistry are: front row: Ronald M. Ferry (second from left), E.J. Cohn (fourth), J.T. Edsall (second from right). with Walter Cannon, headof the Departmentof Physiology, is fifth from left. Second row: J.P. Greenstein (extreme right) T.L. McMeekin, next to him. Fourth row: department secretary AnnaB. Curtiss (second from right). Lastrow: technicians George Greco, John H. Weare, and Muriel H. Blanchard (three, four, and five from left).
1530 of very high electric moment, with positive and negative ionic charges, separated by about 3 A. A series of peptides, as the number of residues increased, would haveincreasing separation of the charges with each added residue; so they could have very highdipole moments indeed. We called these molecules dipolar ions; I now think that ionic dipole would be a better term and will use it here. Jeff Wyman undertook to measure the dielectric constants of amino acids and peptides in water solution, to test thishypothesis. This required a new approach; the existing methods could not handle solutions of such relatively highconductivity as these solutions. Within 2 years he had solved the problem and studied a series of amino acids and peptides with McMeekin. The results spectacularly supported theionic dipole hypothesis; the dielectric constants of these solutions were linear functions of the concentration. A molar solution of an a-aminoacid gave a value 22.5 units higher than that of water; for dipeptides the corresponding value was 70, for tripeptides 113, and so on. Wyman’s interpretation of his data stimulated Lars Onsager to produce the first good theory of the dielectric constants of polar liquids. I took adifferent approach to the ionic dipole hypothesis, by the study of Raman spectra of aqueous solutions of amino acids and related compounds. The characteristic vibrational frequencies of the amino and carboxyl groups in the charged and uncharged states were easy to obtain, from amines and carboxylic acids, and the comparison with isoelectricamino acids immediately showed that both groups were charged. Raman spectra were also valuable for studying many other problems, such as the effects of exchanging deuterium for hydrogen; but these need not be considered here. There were other implications of the ionic dipolar structure ofthe amino acids and peptides. The apparent molar volumes ofa-amino acids in water proved to be some 13 cm3smaller than those of isomeric polar but uncharged compounds; a comparison of glycine and glycolamide was the simplest example. The difference was due to the electrostriction of water moleculesthat were squeezedtightly around the charged groups by the electric fieldof the iondipole. The same mechanism also produced a marked lowering of the heat capacity of the system. These effects, of course, were already well known for ions. McMeekin, with various other members of the laboratory, carried out along seriesof solubility studiesof amino acids in various media, varyingboth the ionic strength and the dielectric constant of the solvent medium; the latter was variedby adding alcohol or acetone. Jack Kirkwood, sometimes in association with Scatchard, developed for dipoles an extension of the Debye-Huckel theory of interionic attractions and studied several modelsfor dipoles, at least one of which gavea very good fit to McMeekin’s data. Kirkwood’s brilliant theoretical analysis was one of the first steps in hisoutstanding career, culminating in the chemistry department at Yale, and ending tragically in his death from cancer at the age of 52.
J. T. Edsall
McMeekin also studied the relative solubility of amino acids in water and organic solvents of lower dielectricconstant, such as ethanol. Nonpolar side chains shifted the relative solubility in water and ethanol according to a simple rule; glycine was some 2,000 times as soluble in water as in ethanol; for alanine the ratio was near 700; for aaminobutyric acid it was near 250. Each added CH2 group altered the ratio by a factor close to three. This was another example of the rule formulated by Isidor Traube in 1891, as a result of his studies of the lowering of the surface tension of waterby members of a homologous series, suchas alcohols or fatty acids, containing a polar end group and a hydrocarbon chain. The concentration in the solution that was required to lower the surface tension by a given amount decreased by a factor close to three for each added CH2 group in the chain. Traube’s work was perhaps the first description of whatwe now knowas hydrophobic interactions. I have written about his life and work elsewhere. I published a small contribution to the hydrophobic effect in 1935. Lookingat other people’s data for molar heat capacities in some homologous series of pure organic liquids, and then at the data for the apparent molar heat capacities of the same compounds in aqueous solution, it was clear that the increment in molar C, per CH2 group (in cal mol”) was far larger in aqueous solution (20-30) than in the organic liquid (5-8). Apparently none of the workers involved had commented on this striking difference. I did not know what itmeant, but the difference was startling, and I called attention to it in a two-page paper in the Journal of the American Chemical Society. In recent years such people as Julian Sturtevant, Ingemar Wadso, Stanley Gill, and Peter Privalov have vastly extended the data and have shown that these striking increases in heat capacity, on exposure of hydrophobic groups to water, as for instance in protein unfolding, represent a general phenomenon. There were ofcourse other studies on what we now call hydrophobic effects in the 1930s, such as the important work of J.A.V. Butler on the enthalpy and entropy changes for aseries ofalcohols dissolved inwater. However our thoughtson these matters were for themost part fragmentary. The first clear and coherent look at these phenomena, in their relationto protein structure and function, did not come until 1959, in Walter Kauzmann’sgreat paper in Vol. 14 of Advances in Protein Chemistry. With this backward look at some of the early days in protein chemistry I conclude these unsystematic reminiscences. With the coming of war in Europe in 1939, and the entry of the United States into the war in 1941, we entered a different era in which the Department of Physical Chemistry was mobilized for scientific service inthe war, like thousands of other laboratories. Much has been written about the blood plasmafractionation project and what it accomplished, in war and later in peace, but thatis another story.
