F. SANGER 



be found that would split between these residues. It was thus 

 necessary to use acid hydrolysis to identify these two disulfide 

 bridges. 



In contrast to that found in neutral solution, the interchange 

 reaction in acid was found to be markedly inhibited by SH 

 compounds. The mechanisms of the two reactions thus appear 

 to be different. Moreover, it was possible to make use of this 

 inhibition to find conditions in which insulin could be hydrolyzed 

 to fairly small peptides in acid without any appreciable inter- 

 change occurring. To simplify the position somewhat the 

 chymotryptic "core" which contained only two of the three 

 cystine residues was studied. In Table V are listed the various 



TABLE V 

 Cysteic Acid Peptides Found in the Oxidized Partial Hydrolyzate of the 



Chymotryptic "core" of Insulin 



CySOsH Gly • lieu • Val • Glu • GIu • CySOsH 



CySOsHGly CySOsHAla 



LeuCySOsH ValCySO^H 



Leu-CySOsH-Gly Ser- Val-CySOsH 



HisLeuCySOaH CySOjH • CySOsH 



His • Leu • CySOsH • Gly CySOsH • GySOsH • Ala 



Glu • CySO sH Glu • CySO 3H • CySO 3H 



Glu • Glu • CySOsH Glu • CySOaH • CySOjH • Ala 



cysteic acid peptides which were present in the hydrolyzate 

 after oxidation. It can be calculated that 95 different cystine 

 peptides would be present in the unoxidized hydrolyzate to give 

 rise to the 16 cysteic acid peptides. Of these 95, 12 contain a 

 half cystine residue (CyS-) and thus provide no information 

 about the distribution of disulfide bonds, 72 contain the group 

 CySCyS and are also of no use, whereas 11 contain a peptide on 

 either side of the disulfide bond and provide the information 

 required. To obtain as little of the peptides with CySCyS as 

 possible, a fairly long period of hydrolysis was necessary, as this 

 group was particularly stable. The conditions eventually 

 chosen were to boil the insulin 45 minutes with 10 iV H2SO4 in 

 30% acetic acid containing 0.005 M thioglycolic acid. 



452 



