F. SANGER 



glycine as the A^-terminal residue; it contained no basic amino 

 acids and no threonine, proHne, or phenylalanine. Fraction B 

 contained a phenylalanine iV-terminal residue and all amino 

 acid found in insulin except for isoleucine. All the basic amino 

 acids were present in this fraction. From a quantitative deter- 

 mination of the A^-terminal residues it was calculated that the 

 molecular weight of fraction A was 2900 and that of fraction B 

 3800. Sulfur estimation indicated that fraction A contained 4 

 cysteic acid residues, which had originated from the cystine 

 residues of insulin, whereas fraction B, the longer chain, con- 

 tained only two. 



Although not fully realized at the time, this oxidation pro- 

 cedure had specifically split the insulin molecule of 50 residues 

 into two chains containing 20 and 30 residues, respectively, and 

 had thus considerably simplified the problem of structure deter- 

 mination. 



From the fact that insulin contained two A^-terminal 

 residues it was suggested that only two polypeptide chains were 

 present. This, however, was not entirely certain, since there 

 might also have been cyclic chains, which would contain no 

 iV-terminal residue. These, however, would be expected to 

 appear after oxidation as a fraction containing no end group. 

 The fact that there was no evidence of any such fraction con- 

 firmed that the glycyl and the phenylalanyl chains were the 

 only ones present in insulin. 



N -Terminal Sequences 



Hitherto the position of only one amino acid in each chain 

 had been determined. These were the A^- terminal residues, 

 which were located by the DNP technique. The method could, 

 however, be extended to the identification of other residues near 

 to this A^- terminal position (16). Thus, whereas complete 

 hydrolysis of the DNP derivative of fraction B gave DNP- 

 phenylalanine, partial hydrolysis gave a mixture of DNP- 

 phenylalanyl peptides, which were fractionated by partition 



438 



