192 EMIL L. SMITH, ROBERT L. HILL AND J. R. KIMMEL [11 



In concluding the present phase of this presentation, it may be noted 

 that the problem of dealing with the tryptic peptides from a protein as 

 large as papain is far from simple. Chromatography by itself has given us 

 very few pure peptides from the tryptic digest of oxidized papain. In fact, 

 we have been badly misled on some occasions in that good stoichiometry 

 has been obtained on amino acid analysis, only to find that several amino 

 end-groups were present as shown by the dinitrophenyl method or that 

 several components could be identified by ionophoresis on paper. 



DEGRADATION OF MERCURIPAPAIN 

 BY LEUCINE AMINOPEPTIDASE 



The isolation of leucine aminopeptidase in essentially homogeneous form 

 and demonstrably free of other peptidases and of proteinases^* has per- 

 mitted investigations of the action of this exopeptidase on polypeptides and 

 proteins. In brief, we have been able to show that the aminopeptidase is 

 restricted in its hydrolytic action to peptide bonds adjacent to a free a-amino 

 group, regardless of the size of the substrate. This has been demonstrated 

 not only with small, synthetic, compounds^^ but also with the oxidized A 

 and B chains of insulin, with zinc-free insulin, with oxidized ribonuclease 

 and a number of other susceptible proteins. 2^" ^^ In no case, was it possible 

 to obtain evidence of a hydrolytic action at any locus other than a peptide 

 bond at the amino-terminal end of the substrate molecule. As an illustra- 

 tion of these findings, it may be noted that the aminopeptidase has been 

 successfully used to determine the sequence of the first six residues at the 

 iV-terminal end of the oxidized B-chain of insulin. ^^ 



The known specificity of the aminopeptidase permits use of the enzyme, 

 not only for sequence studies, but for other types of investigations as well. 

 Differentiation between glutamyl and aspartyl residues from their respec- 

 tive cü-amides is simple since the enzyme acts only on a-peptide or a-amide 

 bonds. Moreover, when an optically active amino acid residue is liberated, 

 it must be of the l configuration, since the aminopeptidase does not release 

 A^-terminal d residues. This specificity also allows the enzyme to be used 

 for resolution of DL-a-amino acid amides. It may be emphasized that we 

 have been unable to find any measurable transferase or synthetic action by 

 the enzyme,^" findings which insure unequivocal results in structural studies. 



The sharp specificity of the aminopeptidase permits studies of the rela- 

 tionship between the structure of the amino-terminal end of a susceptible 

 polypeptide or protein and the biological activity of the substance. The 

 present section is devoted to a study of the action of the aminopeptidase 

 on mercuripapain. 



Active papain cannot be employed as a substrate for the aminopeptidase, 

 since the latter is rapidly digested and inactivated by the proteinase ;^^ how- 

 ever, mercuripapain is essentially inert. It has already been reported briefly ^"^ -^^ 



