Insulin has been extensively studied since it was discovered by Banting and Best in 1921. Early in 1934, Dorothy Crowfoot and John Desmond Bernal obtained the first X-ray diffraction photograph of an enzyme protein: pepsin. In 1935, they took another photograph of a protein hormone: insulin. The chemical structure of protein was unknown until the amino acid sequence of bovine insulin was solved by Fred Sanger and colleagues in 1955. In 1958, the chemical synthesis of bovine insulin started in China through a nationwide collaboration of three institutions: the Institute of Biochemistry in Shanghai, the Institute of Organic Chemistry in Shanghai and Beijing University. The total synthesis of bovine insulin in crystalline form was accomplished in 1965. The success of the synthesis of the first protein in vitro greatly encouraged young researchers in China. Not long afterwards, the project of structural analysis of insulin crystal was carried out in China through the collaboration of the Institute of Biophysics, the Institute of Physics and Beijing University, and succeeded in 1971. In Dorothy Hodgkin's laboratory in Oxford, X-ray diffraction studies of insulin crystals were resumed after about 30 years, and the structure of rhombohedral insulin crystal was solved in 1969. Through insulin research, the Institute of Biophysics in Beijing and the Institute of Biochemistry in Shanghai established scientific collaboration and personal friendship with Dorothy Hodgkin's laboratory in Oxford, and later Guy Dodson's laboratory in York and Tom Blundell's laboratory in London. In 1975, Dorothy Hodgkin wrote a short note, ‘Chinese work on insulin’ in Nature, anticipating closer scientific exchange between the East and the West. In 1982, a bilateral meeting between the Biochemical Societies in the U.K. and China was held in Oxford. Now, the second bilateral meeting held in Shanghai will further promote the collaboration between our two countries.

Introduction

On the occasion of the Centenary of the founding of Biochemical Society, the Joint Sino–U.K. Protein Symposium was held in Shanghai in May 2011. In the present article, I focus on insulin research in China and the U.K., confined to what I have experienced or what I knew personally. Figure 1 is a photograph taken in Oxford where the Biochemical Society–Chinese Biochemical Society Joint Colloquium was held in 1982 as the 600th meeting of the Biochemical Society. The meeting was organized by Rodney Porter and Fred Sanger. The first three presentations all focused on insulin research. The first speaker was Dorothy Hodgkin (née Crowfoot). In the beginning of her presentation entitled ‘Studies on insulin structure and function’, she said, “It seems appropriate to begin this discussion by illustrating first a photograph, now about 50 years old, of two young men whose interests and friendship played a considerable part in bringing this particular audience together: Joseph Needham, before he became passionately interested in China and Chinese science, and John Desmond Bernal, before he took the first X-ray diffraction pictures of pepsin crystals in 1934. Bernal's observations opened the way for the studies we are considering today on insulin, to which our Chinese colleagues have notably contributed.” Joseph Needham was a great friend of Chinese people. During the Second World War, he was the head of the Sino–British Science Cooperation Office in Chongqin, the war capital of China. His secretary and interpreter was Tianqin Cao (Figure 2), a graduate from Yenching University. Through this close collaboration, a long-term friendship was established between them. In 1946, Tianqin Cao went to Cambridge to study muscle proteins in Kenneth Bailey's laboratory. In 1952, he returned to China and continued his research in the Institute of Biochemistry. He had carried out pioneering work on muscle proteins and trained many outstanding workers of protein research. He was also one of the main organizers in the important project of total synthesis of insulin in China. In England, John Desmond Bernal and Dorothy Crowfoot had performed pioneering work in protein crystallography. Early in 1934, they obtained the first X-ray diffraction photograph of an enzyme protein: pepsin [1]. They first noticed that, “it was clearly necessary to avoid alteration of the crystals, and this was effected by drawing them with their mother liquor and without exposure to air into thin capillary tubes of Lindemann glass… …Not only do these measurements confirm such large molecular weights, but they also give considerable information as to the nature of the protein molecules and will certainly give much more when the analysis is pushed further.” In 1935, Dorothy Crowfoot took single crystal X-ray photographs of a protein hormone: insulin [2]. At that time, it was too difficult to solve the structure of a protein with such a high molecular mass until the method of heavy atom isomorphous replacement was worked out by Max Perutz and his co-workers. After about 30 years, the high-resolution structure of porcine insulin crystal was eventually solved in Hodgkin's laboratory in 1969 [3]. The success of the total synthesis of insulin in China in 1965 greatly encouraged the young researchers. In 1967, the structural analysis of insulin crystal was carried out in China through the collaboration of the Institute of Biophysics, the Institute of Physics and Beijing University. In 1971 and 1973, the three-dimensional structure of porcine insulin crystal was solved at 2.5 and 1.8 Å (1 Å=0.1 nm) resolution successively [4]. A postage stamp was issued in China in 1976 in recognition of this achievement (Figure 3). Through insulin research, the Institute of Biophysics in Beijing and the Institute of Biochemistry in Shanghai established scientific collaboration and personal friendship with Dorothy Hodgkin's laboratory in Oxford, and later Guy Dodson's laboratory in York and Tom Blundell's laboratory in London (Figures 4–6). In August 1972, Dorothy Hodgkin visited the Institute of Biophysics in Beijing and the Institute of Biochemistry in Shanghai before she went to Kyoto to join the 9th International Congress of Crystallography. She introduced Chinese work on insulin during the Kyoto meeting. In 1975, Dorothy Hodgkin wrote a short note, ‘Chinese work on insulin’ in Nature, anticipating closer scientific exchange between the East and the West [5].

Photograph taken during the Biochemical Society–Chinese Biochemical Society Joint Colloquium held in 1982 in Oxford

Tianqin Cao and Xide Xie (Cao's wife) entertained Joseph Needham at home in Shanghai

Postage stamp showing the structure of insulin crystal and postcard sent in 1977

Photograph taken in 1977 when Dorothy Hodgkin, Guy Dodson and Eleanor Dodson visited the Institute of Biochemistry in Shanghai

Photograph taken in 1980 when Dorothy Hodgkin and Tom Blundell visited the Institute of Biochemistry in Shanghai

Photograph taken in 1979 when Dorothy Hodgkin, Guy Dodson, Zhizhen Wang and Youmin Feng attended the 2nd International Insulin Symposium in Aachen

Now I concentrate briefly on the total synthesis of bovine insulin in China. This project was proposed by a group of researchers in the Institute of Biochemistry in 1958 after the amino acid sequence of insulin was determined in Sanger's laboratory in 1955. This proposal was apparently influenced by the famous saying of Frederick Engels: “Life is the mode of existence of albuminous bodies, and this mode of existence essentially consists in the constant self-renewal of the chemical constituents of these bodies.” To carry out this project, a nationwide collaboration was established. The Institute of Biochemistry was responsible for the regeneration of native insulin, the synthesis of the B chain and the combination of synthetic A and B chains. The Institute of Organic Chemistry and Beijing University were responsible for the synthesis of the A chain. Before starting the total synthesis of insulin, the following questions must be answered. Can we synthesize the A and B chains separately and combine them to obtain synthetic insulin? Does synthetic insulin possess the native conformation and exhibiting biological activity or does it turn out to be a denatured molecule without any biological activity? Therefore the regeneration of native porcine insulin was studied by a small group in the Institute of Biochemistry. In 1957, Swan [6] reported in Nature the reversible conversion of disulfide bonds into S-sulfonate groups. Using this method, insulin was split into the A and B chains with stable S-sulfonate groups. After purification, the two separated chains were mixed and reduced by a thiol-reactive reagent to convert the S-sulfonate groups into thiol groups. After air oxidation, regenerated insulin with 5–10% insulin activity was obtained. The crude product of regenerated insulin showed 5–10% activity and contained 90–95% impurities. Its purification was a challenge at that time when no sophisticated facilities were available. Using a simple and efficient extraction method, highly purified regenerated insulin was obtained in crystalline form. The crude product was extracted with the upper phase of an acid solution containing equal volumes of secondary butanol and water. The extract was transferred to aqueous solution at mild alkaline pH and dialysed against a solution optimal for insulin crystallization. After storage overnight in the fridge, rhombohedral crystals were obtained [7]. The crystalline regenerated insulin exhibited nearly full insulin activity. The regeneration of insulin activity is similar to the regeneration of ribonuclease activity carried out in Anfinsen's laboratory at about the same time. The regeneration of insulin from two separate chains was much more difficult than the regeneration of ribonuclease from a single chain. The recombination of the A and B chains to form regenerated insulin with 5–10% insulin, and its purification and crystallization to a product with identical crystal form and exhibiting almost full insulin activity demonstrated unequivocally the same principle proposed by Anfinsen, i.e. “the three-dimensional structure of a protein is determined by its primary structure”. After the success of insulin regeneration, the synthesis of the A and B chains was carried out by fragment condensation in solution, which was a long journey of research including the ingenious design of protecting groups and coupling methods. All intermediates must be purified; the quality of synthetic A and B chains must be examined by semi-synthesis with the corresponding native B and A chains. The total synthesis of crystalline bovine insulin in China was achieved in 1965 and published in 1966 [8]. The synthetic insulin with crystalline form identical with that of native insulin and exhibiting almost full biological activity (23 IU/mg) was the first protein ever synthesized in vitro.

Joint Sino–U.K. Protein Symposium: a Meeting to Celebrate the Centenary of the Biochemical Society: A Biochemical Society Focused Meeting held at Shanghai University, Shanghai, China, 5–7 May 2011. Organized by Tom Blundell (Cambridge, U.K.), Zengyi Chang (Peking University, China), Ian Dransfield (Edinburgh, U.K.), Neil Isaacs (Glasgow, U.K.), Glenn King (University of Queensland, Australia), Sheena Radford (Leeds, U.K.), Zihe Rao (Nankai University, China), Yi-Gong Shi (Tsinghua University, China), Chihchen (Zhizhen) Wang (Institute of Biophysics, Chinese Academy of Sciences, China), Jiarui Wu (Shanghai Institute of Biological Sciences, China) and Xian-En Zhang (Ministry of Science and Technology, China). Edited by Zengyi Chang and Neil Isaacs.

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