11] STRUCTURE AND ACTIVITY OF PAPAIN 193 



that the aminopeptidase liberates a large quantity of amino acids from 

 mercuripapain, the extent of degradation depending on the ratio of enzyme 

 to substrate. The data of Table 5 indicate that as much as two thirds of the 

 amino acid residues of mercuripapain can be liberated. What is most strik- 

 ing is that in each case the enzyme, after removal of mercury, still retains 

 its activity towards benzoyl-L-argininamide (Table 5). These results indicate 



Table 5 



DEGRADATION OF MERCURIPAPAIN BY LEUCINE 

 AMINOPEPTIDASE 



Mg++-activated aminopeptidase {E) was allowed to act on mercuripapain iS) for 24 

 hours at the pH indicated. The molar ratio of 5 to £ is indicated. Extent of degradation 

 was estimated by the increment in ninhydrin color expressed as leucine equivalents. 

 Activity was estimated with benzoyl-L-argininamide as substrate as previously described.^ 

 Activity of degraded mercuripapain, after activation, given as per cent of control before 

 digestion. From Hill and Smith.^s 



that the active site of papain must be remote from the TV-terminal region 

 of the molecule. 



In documenting the above observations, it is obviously important to de- 

 monstrate that only amino acids are liberated and that these are derived 

 entirely from the A^-terminal end of mercuripapain, as expected from the 

 specificity of the aminopeptidase and from experience with other polypep- 

 tides and proteins. Secondly, it is desirable to study the residual mercuri- 

 papain itself to determine that the active molecule differs from the original 

 protein. Thirdly, we have attempted to ascertain whether the specificity of 

 the degraded papain is altered. It may be recalled that Perlmann^^ found 

 that the activity of pepsin fragments, obtained by autolysis, differed for 

 protein and peptide substrates as compared with the intact enzyme. 



Free amino groups. As already noted above, the DNP amino acids found 

 after hydrolysis of intact DNP-mercuripapain are one equivalent of DNP- 

 isoleucine and 8 of e-DNP-lysine. Table 6 gives the findings with mercuri- 

 papain hydrolyzed by the aminopeptidase with different numbers of 

 residues released. It is evident that the mercuripapain must be hydrolyzed at 

 Nps 



