HSIEN WU AND THE FIRST THEORY OF PROTEIN DENATURATION (1931)

By JOHN T. EDSALL
Department of Molwular and Callular Blology, Harvard Unlvarrlty, Cambrldge, Marrachurettr 02138

I write to introduce a paper that stands apart from everything else in this volume; it originally appeared in 1931, more than two generations ago. T h e papers that follow it are all by authors who work at the frontiers of the science of today in the great endeavor to solve the central problem of protein folding and unfolding. Hsien Wu, in this paper of long ago, after years of work and thought, was ( I believe) the first to grasp the fundamental relations between the native and the denatured state of protein molecules. Later workers have greatly enriched his picture, but his proposal still stands as a landmark in the field. Wus paper is, I fear, unknown to many biochemists of today; and it should not be forgotten. It appeared, in English, in the Chinese Journal o f Physiology (H. Wu, 1931), and its author, Hsien Wu (1893-1959), was then a Professor in the Peiping Union Medical College in the capital city now known to us as Beijing. The Medical College had been established and financed by the China Medical Board of the Rockefeller Foundation, which watched over it with constant interest, concern, and encouragement. Before commenting on Wus theory of denaturation and its origins, I give a brief account of his life, drawn from the excellent memoir prepared after his death by his wife, Daisy Yen Wu (D. Y. Wu, 1959).
A SHORT ACCOUNT OF THE LIFE AND WORK OF HSIEN Wu Hsien Wu, known by the courtesy name of Tao-min to his Chinese friends, was born in Foochow, Fukien, China, on November 24, 1893. He came of a scholarly family, the second of three children. At the age of 17, he was one of a selected group of students chosen by the Chinese government for a scholarship award to study in the United States. Shortly after the arrival of the group in San Francisco came the overthrow of the Manchu dynasty and its replacement by the Republic of China; but this momentous event did not disturb the planned arrangements for Wus education. Settling in Cambridge at the Massachusetts Institute of Technology, he enrolled at first in the study of naval architecture, but soon shifted
ADVANCES IN PROTEIN CHEMISTRY, Vol. 46

Copyright 8 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

JOHN T. EDSALL

to chemistry as his major field, with a minor in biology, and received his Bachelor of Science degree in 1916. He continued there for one year as a graduate student in organic chemistry, but then moved to Harvard Medical School, where he worked with the famous biochemist Otto Folin. In 1919 a paper based on Wus Ph.D. thesis, A System of Blood Analysis, appeared in the Journal o f Biological Chemistry (Folin and Wu, 1919). It described a new and substantially improved procedure for preparing a protein-free blood filtrate, 10 ml of which sufficed for the determination of nonprotein nitrogen, urea, creatinine, creatine, uric acid, and sugar. This was one of the most influential papers ever published from Folins laboratory. With some supplementary material published a little later, the whole procedure became known as the Folin-Wu methods, which were widely used in biochemical and clinical laboratories for a number of years. Several years ago Harvard Medical School created a Folin-Wu Room, commemorating both men and their work. In the spring of 1920 Wu received an appointment in the newly organized Peiping Union Medical College (PUMC) and returned to China. In 1924 he became Associate Professor and Head of the Department of Biochemistry, and in 1928 became Professor. In 1923 he was one of the three coauthors of a classical study of the distribution of gases, electrolytes, and water between red cells and blood plasma (Van Slyke et al., 1923). In addition to his work on proteins, he did extensive work on nutrition and immunology, and later served on international commissions concerned with nutritional problems. In 1924 he married Daisy Yen, who had become an expert in nutrition by working with Professor H. C. Sherman at Columbia University. Returning to PUMC, she assisted Dr. Wu in his teaching and initiated an analysis of Chinese foods, which later grew into a major project. Hsien and Daisy Wu had five children, one of whom, Ray J. Wu, is Professor of Biochemistry at Cornell University. Hsien Wu continued his active program of teaching and research, and became involved in many other activities. He helped organize the Chinese Physiological Society in 1926, was elected its President, and served on the Editorial Board of the Chinese Journal of Physiology (1926- 194 1). He served as Director of PUMC from 1935 to 1937. In 1937 the Japanese invaders occupied the city of Peiping, but they allowed PUMC to continue independently until 1942, when they took it over also. At that point Wu withdrew, and stayed at his home, an old ducal palace, where, as his wife describes, he devoted his time to reading, writing, practicing Chinese calligraphy, and enjoying his family and his home. He also became more deeply concerned with political, social, and economic problems. Early in 1944 he got word that the Chinese government-in-exile in Chungking wanted him to come there to establish a Nutrition Institute.

FIRST THEORY OF PROTEIN DENATURATION

It was a long and dangerous journey, into Free China, under primitive conditions, by rail, mules, charcoal-burning buses, and on foot; and of course his departure had to be kept secret from the Japanese. On arrival in Chungking he drew up plans for the Nutrition Institute, and was appointed its first Director. The government then sent him to the United States, to study problems of postwar reconstruction in developing the plans for the institute. This involved another long difficult journey; it was over 2 months before he reached Los Angeles. He spent about a year in the United States, visiting many institutes and experts whose work was related to his mission, and returning to Chungking after the Japanese surrender in 1945, to plan the proposed institute in more detail. He returned to Europe and the United States in 1947-1948, a period of turmoil in China as the communist armies steadily gained ground against the Chiang Kai Shek government. He planned to sail home and join his family, who were still at their home in Peiping, but the steamship company twice canceled sailings. As the communist armies advanced on Peiping, in November 1948, Daisy Wu realized that the situation was becoming intolerable for her and the children. They fled to Shanghai, and then to Hong Kong, where they had to wait 4 months before they could get passage to America. He met them when they arrived in San Francisco in July 1949. The Medical College of the University of Alabama gave Wu a visiting professorship, where he taught first-year medical students and carried on research, assisted by his wife, who soon was appointed a research associate. His prime research interest by this time was in nutrition. In October 1952 he suffered a very severe coronary attack, from which he recovered slowly. This led to his resignation from the Medical College in August 1953. He then moved to the Boston area, which he had always called his second home, after the 9 years he had spent at MIT and Harvard Medical School. He settled in Brookline with his wife and children, leading a quieter life than before, reading widely and writing up the work he had done in Alabama, and produced a series of three papers on the excretion patterns in humans of amino acids labeled with 15N. In April 1958 he suffered a second coronary attack, from which he never fully recovered. He died on August 8, 1959, at the Massachusetts General Hospital. Daisy Wus monograph (D. Y. Wu, 1959) contains numerous illuminating tributes to him by those who had known him. HSIEN Wus THEORY OF PROTEIN DENATURATION The reprint of Wus theory of denaturation (H. Wu, 1931), which follows this introduction, is clearly written and, for the most part, speaks for itself. Wu did not propose a theory until he had done many experi-

JOHN T. EDSALL

ments on various forms of denaturation; this paper was number 13 in a series of papers on denaturation. All the previous papers-the first of which had appeared in 1924-were experimental. In the course of these studies he had experimented with almost all the modes of denaturing proteins that he discusses in this theoretical study. He had given a short preliminary account of his theory, 2 years earlier, when he attended the International Congress of Biochemistry in Boston (1929) and an abstract appeared in the Proceedings of the Congress (H. Wu, 1929). We should recall some aspects of the state of knowledge at the time. Evidence for the idea that denaturation can be a reversible process was very recent. I have given a brief discussion on that elsewhere (Edsall, 1979). Anson and Mirsky (1925) had studied the acid denaturation of hemoglobin, which is complete almost instantly around pH 3, and found that, by careful readjustment of the solution to near neutrality, it was possible to get a fairly good yield of crystalline protein with a normal oxygen-binding curve. In 1926 a comprehensive paper appeared on the reversal of heat coagulation of serum albumin, by Mona Spiegel-Adolf, who worked in Paulis laboratory in Vienna (Spiegel-Adolf, 1926). Wu and Lin (1927) were able to get almost complete reversal of the acid denaturation of methemoglobin and deoxyhemoglobin by working carefully with a mildly alkaline solution; and Mirsky and Anson (1929) greatly improved their earlier procedure for hemoglobin. Crucial to Wus theory, in his paper reprinted here, is the concept that a native protein molecule is a highly compact and well-ordered structure, held together primarily by interactions between polar groups in the main chain and in polar side chains. The molecule therefore can be thought of as a sort of submicroscopic crystal, and the forces that hold the native protein molecule together in a defined configuration are the same as those causing the protein molecules to pack together in a macroscopic crystal. When the protein becomes denatured, and the molecule unfolds into a more or less open chain, it can assume an enormous number of possible configurations (or conformations, in the terminology of today) and specificity is lost, although it can be restored by reversal. Wu, like almost all biochemists in 1931, did not yet know about hydrogen bonds, although a number of physical chemists were beginning to be aware of them. I recall a lecture on hydrogen bonds that Linus Pauling gave at Harvard Medical School-in 1933, I think. I remember his emphasizing that he was sure that such bonds played a very important part in the chemistry of life, although he could not yet specify just how they were important. Mirsky and Pauling (1936) presented a paper on protein denaturation, which has been widely known and has had an important influence. It

FIRST THEORY OF PROTEIN DENATURATION

had much in common with Wus presentation, though they did not cite Wus paper as a reference, and almost certainly were unaware of its existence. They were the first to stress the role of hydrogen bonds in maintaining the structure of the native protein. The importance of such bonds has, I think, never been doubted since, although there is still a dispute as to whether they stabilize or destabilize the native protein. As for hydrophobic interactions, their importance did not clearly emerge until the work of Walter Kauzmann (1954, 1959). For current views on these matters, see, for instance, Rose and Wolfenden (1993). Enough of all this, however-perhaps I have written too much. It is time for the reader to pass on to what Wu said himself, more than 60 years ago.

ACKNOWLEDGMENT
I thank Professor Ray J. Wu of Cornell University, Ithaca, New York, for sending me a copy of his mothers memoir on his father, Hsien Wu (Wu, D. Y., 1959), and for helpful comments on an earlier draft of what I have written here.

REFERENCES
Anson, M. L., and Mirsky, A. E. (1925).J. Gen. Physiol. 9, 169-179. Edsall, J. T. (1979). Ann. N.Y. Acad. Sci. 325, 53-74 (see pp. 70-71). Folin, O., and Wu, H. (1919).J. B i d . Chem. 38, 81-110. Kauzmann, W. (1954). In The Mechanism of Enzyme Action (W. D. McElroy and B. Glass, eds.), pp. 70-1 10. Johns Hopkins Press. Baltimore. Kauzmann, W. (1959). Adu. Protein Chem. 14, 1-63. Mirsky, A. E., and Anson, M. L. (1929).J. Gen. Physiol. 13, 133-143. Mirsky, A. E., and Pauling, L. (1936). Proc. Nut. Acad. Sci. W.S.A. 22,439-447. Rose, G. D., and Wolfenden, R. (1993). Annu. Rev. Bzophys. Biomol. Struct. 22, 381-415. Spiegel-Adolf, M. (1926). Biochem. 2. 170, 126-172. Van Slyke, D. D., Wu, H., and McLean, F. C. (1923).J.B i d . Chtm. 56, 765-849. Wu, D. Y. (1959). Hsien Wu 1893-1959: In Loving Memory. Boston. Wu, H. (1929). Am. J . Phystol. 90, 562-563. Wu, H. (1931). ChineseJ. Physiol. 5, 321-344. Wu, H., and Lin, K. H. (1927). ChineseJ. Physzol. 1, 219-234.