Frederick Sanger pioneered the determination of the amino acid sequence and the composition of protein, most notably the hormone insulin, critical to those with diabetes. Knowing the primary structure of a protein led to its chemical synthesis in the laboratory. This had, and still has, far-reaching scientific and medical importance.

The hormone insulin was the first protein molecule for which the complete structure was determined by chemical means. The primary structure of insulin (its amino acid composition and sequence) was the result of the study of primarily one individual, Frederick Sanger. The results of Sanger’s ten years of study were so momentous that he was awarded the Nobel Prize in Chemistry Nobel Prize in Chemistry;Frederick Sanger[Sanger] . [kw]Sanger Determines the Structure of Insulin (1944-1953)
[kw]Structure of Insulin, Sanger Determines the (1944-1953)
[kw]Insulin, Sanger Determines the Structure of (1944-1953)
Insulin
Biochemistry;hormones
Hormones
Insulin
Biochemistry;hormones
Hormones
[g]Europe;1944-1953: Sanger Determines the Structure of Insulin[01070]
[g]United Kingdom;1944-1953: Sanger Determines the Structure of Insulin[01070]
[c]Chemistry;1944-1953: Sanger Determines the Structure of Insulin[01070]
[c]Biology;1944-1953: Sanger Determines the Structure of Insulin[01070]
[c]Health and medicine;1944-1953: Sanger Determines the Structure of Insulin[01070]
[c]Science and technology;1944-1953: Sanger Determines the Structure of Insulin[01070]
Sanger, Frederick

Proteins, one of the four major compounds associated with living cells, serve a variety of functions in living cells. They can serve as hormones, antibodies, transporters, and the like. In living cells, proteins are being degraded and synthesized continuously. The ability to synthesize proteins at a rate faster than their degradation is usually an indication of the well-being of a cell, a tissue, an organ, a life-form. Without some of these proteins or their normal complement, a cell may cease to function.

The building blocks of proteins are nitrogen-containing compounds referred to as amino acids Amino acids . For the most part, only twenty amino acids are found in protein. A single type of protein, insulin, does not need to contain all twenty amino acids. The difference between one protein type and another, insulin from the oxygen transporter hemoglobin, is primarily which amino acids the protein contains and the order (sequence) they are in. The sequence and composition are collectively referred to as the primary structure. Therefore, the primary structure is the signature of an individual protein.

Insulin was chosen as the first protein whose primary structure was to be deciphered because insulin—bovine, ovine, or porcine in source—was available in pure form and was abundant. The necessity of this protein for life was well documented in 1944 when Sanger began his monumental task. Sanger had the necessary tools to bring his dream to fruition. A technique referred to as chromatography was the main tool employed. Chromatography allows the separation and identification of compounds that are similar in chemical form. For example, even though the amino acids are similar in chemical form, there is sufficient difference between them to be separated and identified by chromatography. For this endeavor, paper was the support medium on which the physical separation was achieved.

Another tool available was a method of separating the amino acids chemically in a protein. When a protein is synthesized, the amino acids are joined to one another by a chemical bond—a carbon atom, common to all amino acids. A carbon atom of one amino acid is joined to a nitrogen atom (again common to all amino acids) of the next amino acid, until the protein is assembled. This bond is called the peptide bond. It had been discovered that proteins could be digested (breaking the peptide bond and chemically separating the amino acids) if treated with hydrochloric acid. The longer the exposure to the acid, the more complete the digestion. The acid digestion was completely random in its beginning stages. Complete hydrolysis usually takes from 24 to 48 hours at 110 degrees Celsius in an evacuated system. This period usually will break all peptide bonds. A shorter period of time will yield free amino acids, as well as partially digested products, of varying lengths (differing in the number of amino acids still in the peptide bond).

Acid hydrolysis of insulin revealed the presence of fifty-one amino acids. Of the twenty possible amino acids found in proteins, seventeen were found in the protein. One amino acid—cysteine—was especially important, yet troublesome. Cysteine contains a sulfur atom that can link up with a sulfur atom of a neighboring cysteine, forming a strong bond referred to as a disulfide bond. This can lead to cross-links in a protein. Fortunately, this bond could be broken specifically with performic acid, which did not break the peptide bonds.

Fifty-one amino acids were found in insulin (composition), but the order in which they occurred (sequence) had to be determined, for example, if the fifty-one amino acids were in one continuous chain or if they were in more than one chain. The presence of cysteine with the possibility of cross-links suggested more than one chain, but it was yet to be determined how many.

Another instrumental tool that aided in the sequencing was the chemical 1-fluoro-2,4-dinitrobenzene (FDNB). This chemical reacts with nitrogen-containing groups of amino acids that are not in peptide bonding. Chemical analysis using FDNB found that insulin consists of two chains. The two chains could be separated from each other by chromatography. What remained was the task of sequencing each chain separately and then determining how the two chains were joined. In one type of procedure, Sanger decided to hydrolyze a chain partially and deduce the sequence of the chain by the fragments produced. This is similar to putting together a jigsaw puzzle in the dark while wearing gloves. Hundreds of fragments had to be generated, separated, reacted with FDNB, and then analyzed. The sequences of both chains were attained by this procedure, but it took Sanger about eight years of analysis.

A second procedure was used to try to determine how the two chains were joined. When a protein containing sulfurs of cysteines joined to one another is acid-hydrolyzed, these sulfurs can recombine in a variety of ways. By complex analysis of these products and the use of enzymes, Sanger was able to inhibit some of these reactions and then was able to determine the proper disulfide bonding involved in holding the chains together. With the setting of the disulfide cross-links, Sanger had succeeded in putting it back together again; it took him another two years.

The scientific community was amazed by Sanger’s ten-year effort. For the first time, the primary structure of a protein was known. Soon after Sanger’s 1953 report, the primary structures of other proteins were forthcoming. From the point of technique, the pathway taken for insulin by Sanger now was employed for hemoglobin, myoglobin, lysozyme, and many other proteins. The technique of chromatography was refined by Sanger and others to a point of its being employed for the purification of other proteins, which is the first prerequisite for sequence determination. Additionally, as an offshoot of chromatography, the technique of electrophoresis was employed in protein and nucleic acid research. This is a cornerstone technique for modern-day nucleic acid sequencing.

Frederick Sanger.

(The Nobel Foundation)

Differences in three amino acids in the A-chain of insulin have been the basis for discriminating one species source of insulin from another; more important, these three amino acids have been found to be the reason for the decreased efficacy of bovine and porcine insulins used in humans in the treatment of diabetes mellitus. After prolonged administration, a person may develop an immunological reaction to the hormone. If this occurs, the species of the administered insulin must be changed.

The knowledge of the primary structure of a protein also opened the door to the possibility of its now being synthesized chemically in the laboratory. This had, and still has, far-reaching importance, in both the scientific and medical arenas. It was no longer a dream to be able to synthesize a protein that could be administered clinically in the human form and in pure form. Growth hormone, interferon, factor VIII (missing in hemophiliacs), and many more proteins now are used routinely in a clinical setting in an attempt to save lives or to increase the quality of the lives of individuals for whom they are indicated. The synthesis of these proteins could be accomplished only if their primary structure were known.

As a further consequence, the studies of many protein chemists gave impetus to the studies of DNA (deoxyribonucleic acid Deoxyribonucleic acid ). In simple terms, DNA contains the message for the primary structure of a protein. DNA molecules are sequences of nitrogenous bases, which are translated into the primary structure of a protein. The knowledge of the primary structure and the elucidation of the genetic code offered the medical world a twofold attack. If the primary structure were known, the gene (a sequence of bases in DNA) for the protein could be synthesized and inserted possibly into the DNA of a host cell, such as bacteria, which would then synthesize the protein for humankind. This idea is the foundation of modern-day genetic engineering and of ambitious scientific projects such as the mapping of the humane genome.

Today, proteins used in the treatment of humans originated either from chemical synthesis or from genetic engineering. Protein and nucleic acid chemistry complement each other. Sanger’s work was so momentous because he pioneered the field. Insulin
Biochemistry;hormones
Hormones

• American Diabetes Association. American Diabetes Association Complete Guide to Diabetes. 4th ed. Alexandria, Va.: Author, 2005. A 554-page resource by the leading institution in diabetes-research advocacy and diabetes education. Discusses the “ins and outs of insulin” and includes a CD-ROM.
• Bliss, Michael. The Discovery of Insulin. 3d pbk. ed. Toronto, Ont.: University of Toronto Press, 2000. An excellent examination of the history of insulin’s discovery in 1921.
• Branden, Carl, and John Tooze. Introduction to Protein Structure. 2d ed. New York: Garland, 1999. Covers research into the structure and logic of proteins. Includes illustrations, mostly in color, a bibliography, and an index.
• 
  Chemistry, 1942-1962. River Edge, N.J.: World Scientific, 1999. The Nobel lectures of laureates in chemistry from 1942 to 1962, including Frederick Sanger, who won the award in 1958. Includes laureate biographies.
• Hunkapiller, M. W., J. E. Strickler, and K. J. Wilson. “Contemporary Methodology for Protein Structure.” Science 226 (1984): 304-311. The authors present methods that were developed since 1959, which currently are involved in protein chemistry. Reveals how some later technology would have reduced Sanger’s efforts drastically.
• Lehninger, Albert L. “Proteins: Covalent Structure and Biological Functions.” In Lehninger Principles of Biochemistry, edited by David L. Nelson and Michael M. Cox. 4th ed. New York: W. H. Freeman, 2005. In this chapter, the concept of sequence determination of proteins as well as its importance is presented in a clear and concise fashion.
• Rawn, J. David. “DNA Replication.” In Biochemistry. New York: Harper & Row, 1983. This chapter shows how sequencing of DNA and proteins went hand-in-hand. Discusses Sanger’s second Nobel Prize for his pioneering efforts in the sequencing of nucleic acids.
• Sanger, Frederick. Selected Papers of Frederick Sanger, with Commentaries, edited by Frederick Sanger and Margaret Dowding. River Edge, N.J.: World Scientific, 1996. A comprehensive collection at 662 pages. Includes Sanger’s writings on the sequencing of amino acids. Illustrations, bibliography.
• Sanger, Frederick, and E. O. P. Thompson. “The Amino-Acid Sequence in the Glycyl Chain of Insulin.” Biochemical Journal 15 (February, 1953): 353-374. This article, although lengthy and technical, describes and elaborates on the difficulties encountered by Sanger in the sequence determination.
• Stein, William H., and Stanford Moore. “Chromatography.” In Biophysical Chemistry: Physical Chemistry in the Biological Sciences, Readings from “Scientific American,” edited by Victor A. Bloomfield and Rodney E. Harrington. San Francisco: W. H. Freeman, 1975. Chromatography was one of the major techniques that was employed by Sanger. This article presents this technique in a nontechnical manner.
• Thompson, E. O. P. “The Insulin Molecule.” In The Chemical Basis of Life: An Introduction to Molecular and Cell Biology, Readings from “Scientific American,” edited by Philip C. Hanawalt. San Francisco: W. H. Freeman, 1973. This review article, written in 1955, is a concise review of the time frame during which the structure of insulin was elucidated. Thompson was instrumental in some of Sanger’s findings.
• Whitford, David. Proteins: Structure and Function. Hoboken, N.J.: John Wiley & Sons, 2005. An excellent introduction to the structure and function of proteins for students in biochemistry and related disciplines.

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