STRUCTURE OF INSULIN 



those involving the carboxyl group of aromatic acid residues, as 

 would be expected from studies with synthetic substrates. It 

 may be that this is due to the presence of another enzyme, 

 though such a structure does not occur in natural proteins. 

 Unoxidized insulin, where residue 1 1 was present as CyS, was 

 not attacked in this way. 



Amide Groups 



When insulin is hydrolyzed with acid there are liberated 

 besides the amino acids 6 moles of ammonia, which are present 

 as amide groups on the co-carboxyl groups of some of the glu- 

 tamic and aspartic acid residues. The method of reduction of 

 Chibnall and Rees (2) could be used to determine the distri- 

 bution of the amide groups between these two types of residues, 

 and in this way it was shown that the three aspartic acid residues 

 were all present as amides, whereas three glutamic acids were 

 amidized and the other four have free 7-carboxyl groups. 



In order to locate these amide groups on the individual 

 dicarboxylic acid residues, two methods were used which depend 

 on the fact that when hydrolysis is carried out with enzymes the 

 amide groups remain intact in the peptides. From the iono- 

 phoretic behavior of such peptides it was in general possible to 

 determine whether the carboxyl groups were free to ionize or 

 bound as amides. For instance the peptide containing cysteic 

 and aspartic acids which was obtained from a chymotryptic 

 hydrolyzate of fraction A migrated towards the anode consider- 

 ably more slowly than the CySOsHAsp obtained from an acid 

 hydrolyzate. It must then have been CyS03H-Asp(NH2), 

 with the C- terminal aspartic acid residue amidized. Similarly 

 the GlylleuValGlu obtained by the action of papain migrated 

 as an acid, thus estabhshing that the glutamic acid residue in 

 position A,4 has a free carboxyl group. Positions of the vari- 

 ous amide groups located in this way are indicated in Figure 2. 



A second method was to determine the amide content of 



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