F. SANGER 



the terminal phenylalanine is accounted for in terms of peptides 

 that fit the sequence DNP-Phe-Val-Asp-Glu, and from this it 

 was concluded that there was only one type of phenylalanyl 

 chain. Further support for this was obtained by studying the 

 hydrolysis of peptide B4 under the same conditions that were 

 used for hydrolysis of DNP insulin. If all the terminal phenyl- 

 alanine in insulin is present as DNP-PheValAspGlu, then the 

 yields of Bl, B2, and B3 should be the same from DNP-insulin 

 as from B4. Table II shows that this is in fact the case. 



Besides reacting with the oi-amino group of phenylalanine 

 in fraction B, fluorodinitrobenzene also reacts with the €-amino 

 group of the lysine residue, which gives rise on complete hydrol- 

 ysis to e-DNP-lysine. On partial hydrolysis yellow peptides 

 containing €-DNP-lysine were obtained, and from their structure 

 and yield it could be shown that all the lysine of insulin was 

 present in the sequence ThrProLysAla. 



A similar study on fraction A showed that the only i\^- 

 terminal glycyl sequence was GlylleuVal-GluGlu. 



C-Terminal Residues 



The identification of C-terminal residues is a somewhat 

 more difficult problem than the determination of iV-terminal 

 residues, and no generally applicable method has yet been 

 worked out. 



When insulin was treated with carboxypeptidase, the first 

 amino acid to be liberated in the free form was alanine (12). 

 From the known specificity of this enzyme it was concluded 

 that a free carboxyl group was located on an alanine residue, 

 and the subsequent determination of the sequence of fraction B 

 indicated that it was the C-terminal residue of the phenylalanyl 

 chain. That asparagine was the other C-terminal was shown 

 independently from the complete sequence of the fraction A 

 (19) and from the fact that a small amount of asparagine is also 

 liberated by the action of carboxypeptidase (10). Recently a 

 more general method has been developed by Chibnall and Rees 



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