Bull. Hist. Chem. 17/18 (1995) 1
THE 1993 DEXTER ADDRESS Thomas Burr Osborne and Chemistry
ph S. r t n, Y l Un v r t
I count it an honor to appear before a meeting of the Division of the History of Chemistry. My talk will deal with the place of Thomas Burr Osborne in the histori- cal development of protein chemistry (1). To begin with, I will describe briefly the state of that field in the late 1880s and the circumstances that brought Osborne into it. I will then try to summarize his work in relation to that of his principal contemporaries. Finally, I will speak about Osborne's qualities as a leader of his research group. Osborne was a Connecticut Yankee who spent his entire life in New Haven. He was born there in 1859, went to Yale for his undergraduate and graduate stud- ies, and studied chemistry there with William Gilbert Mixter. He was the only son of a prominent New Ha- ven banker, who wanted young Osborne to join him in the Second National Bank, but at Yale he had become interested in analytical chemistry. A year after he had received his Ph.D., Osborne joined the staff of the Con- necticut Agricultural Experiment Station, located in New Haven, where he headed the Biochemical Laboratory until 1928. He died in New Haven in the following year (2). When Osborne came to the Station in 1886, its di- rector was Samuel William Johnson [1830-1909], whose daughter Osborne married in the same year (3, 4). His first publications dealt with such matters as soil analy- T. B. Osborne sis, but in 1888, at the suggestion of his father-in- law, Over a thirty-year period, beginning in 1862, he turned to the chemistry of plant proteins, and pub- Ritthausen published an extensive series of papers on lished his first paper on the subject in 1891. The stimu- the preparation and characterization of proteins from lus provided by Johnson was a consequence of the fact plant seeds. When he began this work, the only amino that he was an assiduous reader of the European litera- acids thought to be generally present in proteins were ture on agricultural chemistry, and had come to admire glycine, leucine, and tyrosine. Ritthausen added glutamic the contributions of Heinrich Ritthausen [1826-1912] acid and aspartic acid to the list and showed that hy- (5). During the 1850s, Johnson had worked in Leipzig drolysates of proteins which Liebig had considered to and had met Ritthausen at that time. 2 Bull. Hist. Chem. 17/18 (1995)
be identical b t n differed greatly in their content entific work as a crystallographer, had stated in 1883 of these amino acids. Along with others of his time, (8): "You know that the most complex molecules of plant Ritthausen crystallized the seed globulins from several chemistry are the albuminoid substances. You also know plants. The procedures were very simple: a sodium chlo- that these immediate principles have never been obtained ride solution was allowed to cool slowly, or dialyzed in a crystalline state. May one add that apparently they against water, whereupon well-developed crystals ap- cannot crystallize." Pasteur, by that time the great healer, peared. Some physiological chemists considered such apparently did not know of the work of Ritthausen and crystalline proteins to be important. For example, in the others on crystalline proteins from plant seeds, or chose 1887 (first) edition of his textbook, Gustav von Bunge to ignore it. After egg albumin had been crystallized by wrote (6): "The analysis and investigation of the pure Franz Hofmeister [1850-1922] in 1889, the noted crys- protein crystals and the various products of their cleav- tallographer Arthur Wichmann examined them, and age should provide the groundwork for all of physiologi- wrote ten years later (9) that "There is scarcely a crys- cal chemistry." Apparently Johnson shared this view, but talline substance which, like a sponge, soaks up dissolved it should also be noted that his decision to encourage substances as does albumin." And in 1913, the great Osborne to engage in basic research on the chemistry of organic chemist Emil Fischer [1852-1919], of whom I plant proteins reveals vision and courage in the face of shall have more to say shortly, wrote about crystalline the down-to-earth objectives of the Experiment Station, proteins as follows (10): "... the existence of crystals namely to provide reliable chemical analyses of com- does not in itself guarantee chemical individuality, since mercial fertilizers. isomorphous mixtures may be involved, as is frequently At Johnson's suggestion, therefore, Osborne under- the case in mineralogy for the silicates." Indeed, for most took to repeat and extend Ritthausen's studies, and be- of the German organic chemists of Osborne's time, the tween 1888 and 1901 Osborne's chief aim was the iso- proteins were included among the natural products which lation and purification of the proteins of plant seeds. Be- they chose to denote as Schmiere. ginning with oat seeds, from which he obtained crystal- Also, at the turn of the century, leading biochem- line avenin, he proceeded to study the proteins of over ists had turned to the study of proteins as colloids, which 30 different seeds; indeed, bottles containing samples Thomas Graham had defined as noncrystalline and non- of his preparations are still tucked away in the vault of diffusible substances, and they preferred to apply the the Johnson Laboratory at the Experiment Station. Dur- new physical chemistry to the study of adsorption phe- ing this early phase of Osborne's work, his aim was to nomena exhibited by proteins. It would seem, therefore, prepare what he considered to be pure proteins, and his that Osborne chose to disregard prevalent opinion and, principal criterion for purity was a reproducible elemen- as a well-trained analytical chemist, to begin his work tary analysis for carbon, hydrogen, nitrogen, and sulfur. on proteins by single-mindedly pursuing his goal of pu- Accordingly, he set himself the task of checking the dis- rifying them by crystallization and of drawing conclu- cordant reports in the earlier literature on the elemen- sions about their identity or individuality from their el- tary composition of the seed proteins. As he expressed it ementary composition and their solubility properties. in 1892 (7): In 1892, Osborne reported his findings on the crys- talline globulins from six different kinds of seeds - Bra- The fact that these proteid substances can be artifi- zil-nut, oat-kernel, hemp-seed, castor-bean, squash-seed, cially crystallized is not only interesting in itself, but and flax-seed. He concluded that the first two globulins is important as presumably furnishing a means for making preparations of undoubted purity which will are distinct proteins, and different from the other four, afford a sure basis for further study of their proper- which appeared to him to be the same protein. Two years ties. The contradictory statements made by various later, Osborne found the seed globulins from wheat, investigators, not only in regard to the properties and maize, and cotton to have the same elementary compo- composition of these bodies but also in respect to the sition as the four seemingly identical proteins; and he value of the methods of solution and separation which considered the seven kinds of seeds to contain the same have been employed hitherto, render an exact knowl- globulin, which he named "edestin." By 1903, how- edge of all the facts relating to these substances a mat- ever, he was obliged to revise this opinion, but in the ter of the highest scientific and practical importance. mean- time he continued to amass data on many other Osborne's confidence in crystallization as a means of seed proteins, including the alcohol-soluble prolamines preparing pure proteins was not shared by some of his such as zein and gliadin. In those intervening years, im- contemporaries. Thus, Louis Pasteur, who began his sci- portant advances had been made in protein chemistry, Bull. Hist. Chem. 17/18 (1995) and Osborne changed the direction of his research pro- haps best known for his synthesis of polypeptides. In gram accordingly. December 1905, he wrote to his teacher Adolf von The most important of these advances was the ad- Baeyer as follows (13): dition of many amino acids to the list of regular protein On January 6th I will present a lecture at the Chemi- constituents. Those added between 1880 and 1903 in- cal Society summarizing my work on amino acids, cluded the basic amino acids lysine, arginine, and histi- polypeptides and proteins, and then early next year I dine , as well as phenylalanine, cystine, alanine, valine, will publish the collected papers in the form of a book. isoleucine, proline, hydroxyproline, and tryptophan (11). The material has grown splendidly and there is much In particular, the finding that the basic amino acids form detail in it. Recently I have also prepared the first sparingly-soluble salts with phosphotungstic acid led crystalline hexapeptide and hope to obtain a match- Walter Hausmann, a student in Hofmeister's laboratory, ing octapeptide before Christmas. Then we should to develop in 1900 a method for the determination of be close to the albumoses....My entire yearning is directed toward the first synthetic enzyme. If its the partition of the nitrogen in acid hydrolysates of pro- preparation falls into my lap with the synthesis of a teins among the so-called ammonia-nitrogen, basic-ni- natural protein material, I will consider my mission trogen, and nonbasic nitrogen fractions. Osborne seized fulfilled. upon the Hausmann method, and in 1903 he reported that (12): Although the accounts of Fischer's lecture in newspa- pers and in popular science journals encouraged the We have found by its use that some of our prepara- belief that the preparation of synthetic proteins was tions from different seeds which were so nearly alike around the corner, by 1910 the enormous effort of his in composition and reaction that no difference could assistants had produced much less than he had hoped be detected between them sufficient to warrant the conclusion that they were not the same chemical in- for, and his disappointment may be inferred from the dividual, yield such different proportions of nitrogen fact that after that date there were no further experi- in the several forms of binding that there can be no mental papers on peptide synthesis from his laboratory longer any doubt that they are distinctly different sub- (14). stances. On the other hand, many preparations of dif- In Fischer's ester method, the mixture of amino ac- ferent origin, which we have heretofore considered ids in an acid hydrolysate of a protein was esterified to be identical, have yielded the same proportion of with ethanol, alkali was added to generate the free es- the different forms of nitrogen and consequently our ters, which were then extracted with ether. The ether former opinion respecting the identity of these pro- extract was concentrated and subjected to fractional dis- tein preparations is very greatly strengthened. tillation under reduced pressure, and the esters in the The next step in the development of Osborne's research individual fractions were converted to free amino ac- program was his acceptance, in 1906, of the necessity ids, which were crystallized, weighed, and character- of determining the amino acid composition of protein ized. It surely must have been clear from the start that hydrolysates by means of the methods developed by this method was not likely to give reliable quantitative Albrecht Kossel and Emil Fischer. In 1900, Kossel data for the amino acid composition of proteins. Never- [1853-1927] had introduced a procedure for the quanti- theless, many protein preparations were analyzed in this tative estimation of the three basic amino acids, and in way in Fischer's laboratory; and, apart from demonstrat- the following year Fischer described his so-called ester ing the general occurrence of amino acids such as ala- method for the separation of other amino acids present nine or phenylalanine, three new protein constituents in acid hydrolysates of proteins. Because Fischer's name were found: proline, hydroxyproline, and figures so prominently in the history of protein chemis- diaminotrioxydodecanoic acid. Much of this work was try, I digress briefly from the account of Osborne's work. done by Emil Abderhalden [1877-1950], who had come By 1906, Fischer was widely regarded as the lead- to Fischer's laboratory in 1902, after receiving his ing organic chemist of his time. He had received the Dr.med. degree at Basle. Two years later, in a letter to a Nobel Prize for Chemistry in 1902 for his outstanding Berlin colleague, Fischer wrote (15): achievements in the synthesis of sugars and purines; and, Because of his unusual capacity for work, in a short soon after entering the protein field in 1899, he had ini- time Abderhalden has become so adept in the diffi- tiated an ambitious program to effect the synthesis of cult methods of organic chemistry that I was able to proteins. Apart from his lock-and-key analogy to de- accept him last fall as a collaborator in my private scribe the specificity of enzyme action, Fischer is per- laboratory. I note that I had not dared to do this be- 4 Bull. Hist. Chem. 17/18 (1995)
fore with a medical man. He is a good observer, and bj t t t r r r x n t n th n th t n is an enemy of all superfluous hypotheses. Regretta- d t d n h r l b r t r , nd pr v d t r tl bly, biological chemistry is that part of our science in ( . In th nn t n I nn t f rb r fr t n which imprecise and incomplete experiments are of- p fr l tt r fr h r t Abd rh ld n n ten heavily padded with the dazzling ornamentation 2 ( 8 : of so-called ingenious reflections to producepretentious treatises. For this reason, people I consider it likely that because of their greater wealth like Abderhalden are needed. the Americans will beat us in several fields, and I have expressed this opinion at every opportunity. h p n n l d h r t t rn v r t Abd rh ld n However, we can withstand this competition for a th n f p t M. . t d nt h fl d t time because of our greater inventiveness and more h r l b r t r t th t t , nd t f th distinguished individual achieve- ments. That the r d nd r Abd rh ld n d r t n n th ppl gentlemen in America are also rather presumptuous t n f th t r th d t th n l f r t v r t is nothing new to me, but one can defend oneself f pr t n pr p r t n , n l d n pl nt pr t n h against this at a suitable opportunity. As soon as I d t n nd l d n. v r, h r n t l find the time, I will discuss this question in a retro- nt f Abd rh ld n h l t l nt pr v d t b n spect on chemical research on proteins during the past rr t, f r h f h r b th b r f ten years. h r r p nd n l t r r n nd p nd nt Alth h h n n t nt n d, O b rn th t nv t t r pr v d t b rr pr d bl . In p rt l r, l l nd d t f r h r d pl r , h th h r bl d t thdr d n tr x d d n l d n pr t n h t n th Un t d St t t th t t . d fr h l t f pr t n n d . O b rn ntr b t n t th n l t l h I n r t rn t O b rn r n pr t n . tr f pr t n p rh p b t b ll tr t d b n 06, h h d b n t r v f n n l pp rt fr f bl , t n fr h r v rt l n 0 ( . th C rn In t t t n f W h n t n. h r nt n h f r t l n f n b r nt n th d t ( r bl d h t h r r t nt nd t p r h p p r 00 f pr t n r p rt d f r zein b tth n n nt f r th pr p r t n f th z bl nt f pr 8 2, th nd l n th d t n 0 f n t n t n th n n d d f r th n l f th r n d h d th h r t r th d, nd th th rd l p t n. O b rn ppl d th th d f K l n the values v n b K l nd K t h r n 00 nd h r t th n l f v r l d pr t n nd f r t r n nd th thr basic amino d . h f rth l d th r pr d r t t t th nt nt f nd f fth l n give the values reported from h p n nt tr pt ph n r r , h h r O b rn l b r t r n 06 nd 0, r p t v l . d tr d p n d h dr l . 08, th n r In dd t n t th ntr b t n f K l, t d l d O b rn t r v f rth r h rl r v nn, h r, nd Abd rh ld n t th n l t l b t th h l nd v d l t f th pr t n h h d h tr f pr t n , th r was another r f d p r f d ( 6 : v l p nt h h nfl n d th r f O b rn r r h. h f r t th d v r n 0 b Ott W r n ll past the time when agreement in solubility, ultimate composition and color reactions C hnh [ 8 ] of the enzymatic conversion of are to be accepted as evidence of the identity of two peptones t n d b th nt t n l . preparations of protein....On the basis that agreement f r th t t , n ph l t b l v d th t, n th in ultimate composition affords no evidence of iden- t b l t l z t n f f d pr t n , th p pt n tity of two similar proteins, but that distinct and con- f r d b th t n f p p n nd tr p n r t n p stant differences in composition are conclusive evi- t th nt t n l ll nd nv rt d th r nt bl d pr dence that they are not alike, I ... have since subjected t n . h n xt bl t th d tr n fr Ott them to careful comparison in respect to their physi- [ 8 6 ], h h d n 02 th t cal properties and the proportion of their decomposi- pl t l d t d (p pt n fr p n r t pr t n n tion products, so that those which are alike in their more apparent characters have been still further dis- r pl nt t pr t n n th n l d t. O b rn p tinguished from one another. p r t h v r n z d t n th p rt n f th f nd n , f r n 0 h r t (20 : " h n l... n Ev n th h th f th h r t r th d h d nth z pr t n fr xt r f th r t ll z bl r v l d n d ff r n n th d pr t n , pr d t pr d d b th d p t n f pr t n ." O b rn , ll tr n d n l t l h t, d d n t v r, h d d n t p r th n n f th pt th l t t n f th th d, b t pr d d t d nt l 0 , h n h nd f tt n d t M nd l Bull. Hist. Chem. 17/18 (1995) 5
Table 1. Products of the Hydrolysis of Zein
Component Ritthausen Langstein Kossel & Osborne Osborne (1872) (1903) Kutscher & Clapp & Jones (1900) (1906) (1910)
Glycine 0.00 0.00 0.00 Alanine 0.50 2.23 8.98 Valine 0.29 Leucine 17.25 11.25 18.60 17.95 Proline 1.49 6.53 9.01 Phenylalanine 6.96 4.87 6.23 Aspartic acid 1.43 1.04 1.41 1.73 Glutamic acid 10.00 11.78 18.28 26.17 Serine 0.57 1.00 Tyrosine 3.20 10.06 3.55 3.55 Arginine 1.82 1.16 1.35 Histidine 0.81 0.43 0.82 Lysine 0.00 0.00 0.00 Tryptophan 0.00 0.00 Ammonia 2.56 3.61 3.64
61.53 80.43
[1872-1935] initiated their succeeded Chittenden as famous joint studies on ani- head of the Yale laboratory mal nutrition. In the mean- of physiological chemistry time, Frederick Gowland (22, 23). An outstanding Hopkins [1861-1947], the teacher, Mendel made that discoverer of tryptophan, laboratory the principal had noticed Osborne's report seedbed for the next gen- that the seed protein zein eration of American bio- lacked this amino acid. In chemists. His collaboration 1906, Hopkins and Edith with Osborne in the field of Gertrude Willcock [1879- animal nutrition lasted 1953] published a paper (21) nearly twenty years and showing that young mice produced more than one fed zein as a sole source of hundred joint papers, with protein did more poorly than special emphasis on the so- comparable animals to called indispensable amino whose diet tryptophan had acids and on vitamins. In been added. this work, Osborne's highly A few words about purified protein prepara- Mendel and his relationship tions played a decisive role. to Osborne. Mendel re- Among Mendel's students ceived his Ph.D. at Yale in at that time was William 1893 for work with Russell Cumming Rose [1887- Henry Chittenden [1856- 1985], who later discovered 1943] , and ten years later he threonine during the course L.13. Mendel 6 Bull. Hist. Chem. 17/18 (1995) of his sustained nutritional studies along the lines initi- Vickery - may be said to have achieved scientific dis- ated by Osborne and Mendel (24). tinction. A Canadian who had come to Yale as an 1851 Osborne collaborated to a lesser degree with other Exhibition Scholar, Vickery had begun graduate work American scientists, for example Francis Gano Benedict with Johnson, who recommended him to Osborne. In [1870-1957] and Donald Dexter Van Slyke [1883-1971], 1928, "Vic" (as he was known to his friends) succeeded but the most fruitful of these additional joint efforts was Osborne as head of the Biochemical Laboratory at the the one with noted pathologist Harry Gideon Wells Experiment Station, and in the years that followed he [1875-1943]. Wells had worked in Fischer's laboratory instituted a fruitful program of research on the metabo- with Abderhalden, and his interest in protein chemistry lism of leaves (26, 27). Except for Breese Jones, who led him to examine the specificity of the anaphylactic continued to work productively on proteins after he left response of sensitized guinea pigs to the injection of Osborne, none of the others listed in Table 2 appear to purified seed proteins supplied by Osborne. These ex- have made a significant mark in the scientific litera- periments, reported in 1911, revealed further cases of ture. I should note, however, that in 1913 Frederick Heyl the individuality of proteins previously thought to be became the first research director at Upjohn and during identical (25). the 1930s he initiated that company's pioneering pro- To summarize briefly, the most important features gram on steroid hormones (28). of Osborne's research until about 1915 were succes- Osborne attracted few post-doctoral guests, but sively the purification of seed proteins, the amino acid among them was Edwin Cohn later[1892-1953], who analysis of these proteins, and their use for studies of was in New Haven in 1917. At Harvard, Cohn led a animal nutrition and immunological specificity. After- research group which made many important contribu- ward, Osborne and Mendel were led increasingly into tions to the study of the physical chemistry of proteins such areas as the vitamin content of various foods and (29). Only one post-doctoral associate came to the some medical aspects of nutrition. Osborne laboratory from abroad. He was Albert Charles I turn now to Osborne's research group. At any Chibnall [1894-1988], who had received his Ph.D. in given time, it was quite small and composed almost 1921 at Imperial College London for work on leaf pro- entirely of Yale graduates (see Table 2). It seems that teins. When Chibnall arrived in New Haven, Osborne when one of his assistants was about to leave, Osborne was still in Vermont for his summer vacation - the par- would ask a professor in the Yale Chemistry Depart- tridge season had not yet ended - so Chibnall made con- ment (usually the organic chemist Treat B. Johnson) to tact with Mendel, who impressed him greatly. Vickery recommend someone. Only one of these men - Hubert later recalled that (30):
Table 2. Osborne's Research Assistants
1891-92 Voorhees, Clark Greenwood [1871-1933] Yale Ph.B. 1891 1894-1900 Campbell, George Flavius [1870-1902] Yale Ph.B. 1892 1901-07 Harris, Isaac Faust [1879-1953] Yale Ph.D. 1915 1906 Gilbert, Ralph Davis [1878-1919] Yale Ph.D. 1904 1906-08 Clapp, Samuel Hopkins [1876-1952] Yale B.A.; Ph.D. 1908 1906-09 Brautlecht, Charles Andrew [1881-1964] Yale Ph .B .; Ph.D. 1912 1908 Heyl, Frederick William [1885-1968] Yale Ph.B.; Ph.D. 1908 1908-28 Leavenworth, Charles Stanley [1879-1948] Yale Ph.B. 1902 1908-10 Jones, David Brees [1879-1954] Yale Ph.D. 1910 1909-10 Liddle, Leonard Merritt [1885-1920] Yale Ph.D. 1909 1910-11 Guest, Herbert Hartley [1884-1956] Yale Ph.B.; Ph.D. 1912 1916-23 Wakeman, Alfred John [1865-1956] Yale Ph.B. 1887 1920-28 Nolan, Owen L. [1888-1958] 1921-28 Vickery, Hubert Bradford [1893-1978] Yale Ph.D. 1922 1924-28 Nolan, Laurence S. [1890-1984] Bull. Hist. Chem. 17/18 (1995) 7
When Osborne returned a few weeks later, I told him spicuous indifference to matters which lay outside of this Englishman who wanted to join us. Osborne the well defined boundary lines of his sympathies. was very busy that morning, and my tale was greeted with a succession of grunts and finally, 'Well, is he In a later memoir, Chibnall noted (32): any good?" Assured on this point, he finally said, "All I was surprised to find how narrow his interests were. right, bring him out." Chibnall succeeded in charm- Almost as soon as I came into touch with him I was ing Osborne within the hour, and I was instructed to to learn, to my surprise, that plant physiology made install him in the laboratory at once. As soon as no appeal to him at all. Osborne saw his command of technique, his original approach, and his industry, he happily turned over I have quoted these recollections about Chibnall's as- all of the work on leaf proteins to him. sociation with Osborne for several reasons. The most To this report should be added Chibnall's own later rec- important is that they reveal something of Osborne's ollections (31): style of leadership in his latter years, especially in his ability to recognize scientific talent, as was also evi- I had been warned by Mendel of [Osborne's] ner- dent in his treatment of Vickery. They also confirm the vous temperament, and the possibility that he might impression that Osborne was a man of limited scien- tific outlook. To these reasons I must add an obligation to pay tribute to Chibnall's role in the development of modern protein chemistry (33). In his later research on proteins, as professor at Imperial College from 1929 until 1943, and then as the successor of Hopkins at Cambridge un- til 1949, Chibnall followed the trail charted by Kossel, Fischer, and Osborne. By about 1940, however, with the advent of the chromatographic method introduced by Martin and Synge, Chibnall had begun to see the demise of that approach. At Cambridge, he suggested to a young post-doctoral student named Frederick Sanger that fluorodintrobenzene might be a good re- agent for the determination of the amino-terminal groups of proteins, and that insulin (whose amino acid compo- sition Chibnall's group had determined) might be a good protein to start with. The rest is well-known history. The analytical chemistry of proteins, begun during the 1830s by Gerrit Mulder and Justus von Liebig, was completed during the 1950s by Sanger and by Stanford Moore and Will- iam Stein. In this transmission of a chemical heritage, the role of Thomas Burr Osborne deserves to be remem- bered.
A. C. Chibnall REFERENCES AND NOTES be taciturn when we met, but he greeted me cordially and in a very short while I felt quite at ease. I think I 1. This paper is a revised version of one which appeared in touched a chord to which his nature readily re- Perspectives in Biochemical and Genetic Regulation of sponded, for in our first talk I mentioned my home Photosynthesis (I. Zelitch, Ed.), Alan R. Liss, Inc., New background, and he recognized in me someone who York, N.Y., pp. 1-16, based on my lecture at the Con- had taken the same path as himself, embracing sci- necticut Agricultural Experiment Station on 5 April 1989. ence in spite of family efforts to divert him to more I am grateful to the Wiley-Liss Division of John Wiley practical pursuits. As I got to know him better I & Sons and to Dr. Israel Zelitch for permission to use learned to appreciate the warmth of his interest in that material in the preparation of this paper. things that he cared for, and the scarcely less con- 2, H. B. Vickery, "Thomas Burr Osborne," Biogr. Mem. Natl. Acad, Sci. USA, 1931, 4, 261-304.
:4 8 Bull. Hist. Chem. 17/18 (1995)
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