STRUCTURE OF INSULIN 



was catalyzed by — SH compounds and probably is due to initial 

 hydrolysis of the disulfide and a subsequent chain reaction 

 catalyzed by the — SH compound so produced : 



R1SSR2 """ ) RiS- + R2SOH 

 RiS- + R3SSR4 > R1SSR3 + R4S- etc. 



At pH 7 to 8 the reaction is not very rapid, so it was possible to 

 hydrolyze insulin with enzymes without any extensive mixing up 

 of the disulfide bonds. In this way the position of one of the 

 bridges could be found. Thus for instance the soluble material 

 from a chymotryptic hydrolyzate was subjected to ionophoresis. 

 The cystine peptides were identified by treating a strip with 

 cyanide-nitroprusside reagent (23), and the bands were oxidized 

 and again fractionated by ionophoresis. From one such cystine 

 peptide two cysteic acid peptides were produced with the 

 following amino acid composition : 



[CySOsH, Asp] 

 [CySOjH, Glu, Gly, Val, Leu, Phe, Arg] 



These peptides had been found before in the chymotryptic 

 hydrolyzates of oxidized fractions and were CyS03HAsp(NH2) 

 from the A chain, LeuValCySOgHGlyGluArgGlyPhePhe 

 from the B chain. The structure of the cystine peptide was thus : 



CyAsp(NH2) 



S 



Leu • Val • Gy • Gly • Glu • Arg • Gly • Phe • Phe 



establishing the presence of a disulfide bridge between positions 

 A20 and Bl 9 in insulin. The locadon of the same — S — S — bond 

 was confirmed by the structure of a number of other peptides 

 from chymotryptic and peptic hydrolyzates. The remaining 

 two cystine residues were present in the insoluble material 

 (core) from the chymotryptic hydrolyzate and no enzyme could 



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