The Amino-acid Sequence in the Phenylalanyl Chain of Insulin 1. THE IDENTIFICATION OF LOWER PEPTIDES FROM PARTIAL HYDROLYSATES By F. SANGER (Beit Memorial Fellow)

When insulin is oxidized with performic acid, the -S-Sbridges of the cystine residues are broken by conversion to -SO3H groups (Sanger, 1949 a) and the molecule is split into its separate polypeptide chains. From the oxidized insulin two fractions could be isolated: an acidic fraction A, which contained only glycyl N-terminal residues (see below) and no basic amino-acids, and a basic fraction B, having phenylalanyl N-terminal residues. These appeared to be the only significant fractions present. From a study of the partial hydrolysis products of the dinitrophenyl (DNP) derivatives of the two fractions it was possible to determine the sequence of the amino-acids adjoining the N-terminal residues and adjoining the lysine residues (Sanger, 1949 b). In the case of fraction B the terminal sequence was shown to be Phe . Val . Asp. Glu and the lysine residues were present in the sequence Thr. Pro. Lys Ala (abbreviations for amino-acids are given in Table 1 below). No DNP-peptides were present which did not fit into these sequences, and from an estimation of the yields of the DNP-peptides produced on partial hydrolysis of DNP-insulin it was concluded that all the N-terminal phenylalanyl residues of insulin and all the lysine residues were present in the above two sequences respectively, and hence that there was only one type of phenylalanyl polypeptide chain in insulin. Similar though rather less clear-cut results were obtained for fraction A. Assuming a molecular weight of 12,000, it was concluded from these experiments that insulin is builtup oftwo identical phenylalanyl polypeptide chains and two identical glycyl chains, these four chains being joined together by six -S-Sbridges. Various possible structures for the molecule were suggested (Sanger, 1949c). The results with the terminal peptides gave a preliminary indication that the fractions A and B are both relatively homogeneous preparations of molecules having a single polypeptide chain and containing approximately 20 and 30 amino-acid residues respectively. It thus seemed that an investigation of the smaller peptides produced on