Sanger's Sequencing of Insulin

In the late 1940s, proteins were known to be long chains of amino acids, but nobody knew the order of the amino acids in any of them, or whether each protein had a single fixed sequence. The most discussed theory, from Max Bergmann and Carl Niemann, held that each amino acid recurred at regular intervals along the chain; at the other extreme was the idea that a pure protein was a random mixture of similar molecules. Frederick Sanger, working in the Department of Biochemistry at Cambridge, set out to answer the question by reading the complete sequence of a protein small enough to be tractable. Insulin was a practical choice: Albert Chibnall's group had already analyzed its amino acid composition in detail, and it lacked two common amino acids, tryptophan and methionine.

The technical obstacle was that no method existed to read a protein's sequence directly. Sanger developed one. He introduced 2,4-dinitrofluorobenzene, later called Sanger's reagent, to label the free amino terminus of a peptide chain, which could then be identified after acid hydrolysis. By cleaving insulin with different enzymes and acids at different positions, generating overlapping fragments, and painstakingly mapping where each fragment fell in the overall chain, he assembled the full sequence of both chains over roughly ten years of work. The A chain had 21 residues; the B chain had 30. Two disulfide bonds connected them at specific positions, with a third within the A chain itself.

The paper describing the complete structure appeared in the Biochemical Journal in 1955. Sanger concluded that proteins are definite chemical substances, with one particular amino acid at each position in the chain. The insulin chains also showed no repeating pattern, which ruled out the Bergmann and Niemann theory. If sequence was fixed, it had to be encoded somewhere in the cell, and whatever stored genetic information had to specify an exact linear order.

Watson and Crick had published the double helix structure two years earlier, in 1953. Sanger's result provided the complementary constraint: if DNA was the hereditary molecule, it needed to encode specific sequences, which the base-pair template mechanism could accomplish. The two discoveries together made the genetic code a solvable problem, one that Crick, Brenner, Nirenberg, and others solved by the mid-1960s. Sanger received the Nobel Prize in Chemistry in 1958 for the insulin work.

After insulin, Sanger developed further methods for studying proteins and the active sites of enzymes, and around 1960 he turned to RNA and DNA. In 1977 he and his colleagues published the chain-terminating (dideoxy) method of DNA sequencing. The Human Genome Project used Sanger sequencing, after improving and automating it. Sanger shared the 1980 Nobel Prize in Chemistry for that work, becoming the first person to win the chemistry prize twice; K. Barry Sharpless became the second in 2022.