STRUCTURE OF INSULIN 



(Arg (2) ; Thr, Pro, Lys (3) ; Tyr (4)) are absent. It was thus 

 established that sequence 5 was combined to the A^'-terminal 

 sequence through a GlySer Hnkage. That no other residue 

 could come between the glycine and serine residues was also 

 indirectly demonstrated. From the results with peptic and 

 chymotryptic hydrolyzates it was possible to deduce the three 

 sequences shown in Table IV. 



TABLE IV 



Sequences in Fraction B 



When fraction B was treated with trypsin, a peptide 

 (Bt2) was obtained with glycyl iV-terminal residue and the amino 

 acids [Tyr, Phe, Thr, Pro, Lys]. The last three are found only 

 in sequence 3 (Table IV). The phenylalanine present cannot 

 be the one that is in the TV-terminal position of fraction B, 

 since it is not A^-terminal in peptide Bt2. It must therefore repre- 

 sent the two phenylalanine residues from the C-terminal group of 

 sequence 2 (Table IV), thus establishing that sequence 2 is 

 joined to sequence 3 and that the structure of fraction B is as 

 shown in Figure 2. 



Besides establishing the structure, the study of enzymic 

 hydrolyzates provided considerable information about the spec- 

 ificity of the proteolytic enzymes used. The main sites of action 

 of these enzymes are shown in Figure 2. Trypsin and chymo- 

 trypsin were found to be highly specific and to split only those 

 types of bonds which earlier work with synthetic substrates had 

 indicated would be susceptible. Pepsin, on the other hand, 

 was found to have a much wider specificity which it was diffi- 

 cult to define in terms of the amino acids involved in the sus- 

 ceptible bonds. It seems possible that the specificity of pepsin 

 may not in fact be determined in this way, but rather that the 



445 



