Insulin oxidation with performic acid

These results indicate that insulin is built up of four open polypeptide chains, which seem to be held together by —S—S— bridges of cystine. These are present to the extent of six residues per molecule. By oxidation with performic acid (p. 27) it was possible to split these bridges and thus to liberate the separate peptide chains (Sanger, 1949a). The oxidized insulin was fractionated by precipitation methods and two main fractions were obtained, which appeared to represent the whole of the oxidized insulin. The properties of these two fractions, designated A (acidic) and B (basic), are summarized in Table VIII. The most probable composition of fraction A is,... 

Probably the most important achievement in insulin research was the determination of its primary structure by Frederick Sanger and his associates in Cambridge, England. In order to elucidate the amino acid sequence of the hormone it was necessary to separate the two chains constituting the molecule. This was accomplished by oxidation with performic acid. This operation cleaved the three disulfide bridges by converting each cystine to two cysteic acid residues ... 

Disulfide splitting of insulin by oxidation with performic acid... 

Insulin consists of two different polypeptide chains held together by —S—S— bridges. They can be separated by oxidation with performic acid. Similarly, ribonuclease consists of 124 amino acids and —S—S— bridges which can also be separated. 

The A and B peptide chains in insulin are linked through disulfide bridges. Their presence was suspected from the change in molecular weight which followed the reduction of insulin. For quantitative analyses the S-S bridges had to be broken. Sanger, following the approach used by Toennies and Homiller (1942), oxidized the protein with performic acid, so that the half-cystines were converted to cysteic acid. After oxidation, insulin could be separated into its A and B chains, the A peptide with 20 amino acid residues and the B with 30. 

Thiazolidine residues have also been introduced into protein in other ways. Treatment of insulin in aqueous solution with the hydroxy-succinimide ester of 3-formyl-2,2-dimethyl-L-thiazolidine-4-carboxylic acid (353) yields three products, separable by chromatography. They are characterized, by end-group analysis, electrophoresis, and amino-acid analysis of the performic acid-oxidized chains, as monosubstituted derivatives (354) formed by interaction at each of the free amino-groups of... 

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