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Sanger Was the First to Determine the Sequence of a Polypeptide

المؤلف:  Peter J. Kennelly, Kathleen M. Botham, Owen P. McGuinness, Victor W. Rodwell, P. Anthony Weil

المصدر:  Harpers Illustrated Biochemistry

الجزء والصفحة:  32nd edition.p27-28

2026-03-09

472

While scientists found it relatively simple to determine the amino acid composition of purified proteins, the proportion of each type of amino acid in the polypeptide, deriving the order or sequence of the amino acids proved difficult. The first protein to have its sequence determined was the peptide hormone insulin, an achievement that earned Frederick Sanger a Nobel Prize in 1958. Mature insulin consists of the 21-residue A chain and the 30-residue B chain linked by disulfide bonds. Sanger reduced the disulfide bonds (see Figure 1), separated the A and B chains, and cleaved each chain into smaller and smaller peptides using the proteolytic enzymes trypsin, chymotrypsin, and pepsin followed by incubation with hydro chloric acid. Each peptide in the mixture was isolated, treated with 1-fluoro-2,4-dinitrobenzene (Sanger reagent), and their amino acid composition determined. Sanger reagent reacts with the exposed α-amino groups of the amino-terminal residues, allowing the amino-terminal amino acid of each peptide to be identified. The ε-amino group of lysine also reacts with Sanger reagent; but since an amino-terminal lysine reacts with 2 molecules of Sanger reagent, it is readily distinguished from a lysine from the interior of a peptide. Working from di- and tripeptides up through progressively larger fragments, Sanger was able to reconstruct the complete sequence of insulin. Sanger would later develop the dideoxy method for sequencing DNA, an accomplishment for which he was awarded a second Nobel Prize in 1980.

Fig1. Oxidative cleavage of adjacent polypeptide chains linked by disulfide bonds (highlighted in blue) by performic acid (left) or reductive cleavage by β-mercaptoethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively.