11] STRUCTURE AND ACTIVITY OF PAPAIN 191 



Table 4 



SOME PURE PEPTIDES ISOLATED FROM A TRYPTIC DIGEST 

 OF OXIDIZED PAPAIN 



are being made to resolve these mixtures by other methods. For example. 

 Fraction 10a has been isolated from Fraction 10 plus 11 by ionophoresis 

 on paper and two peptides (8a and %b) have been separated by ionophoresis 

 from Fraction 8, 



The discussion, thus far, has been concerned with the acid-soluble pep- 

 tides resulting from tryptic digestion of oxidized papain. The insoluble resi- 

 due, which is more difficult to fractionate by chromatography, has not been 

 completely resolved. This crude fraction is of great interest since it appears 

 to contain the sulfhydryl group which reacts with iodoacetamide. 



A sample of papain has been treated with iodoacetamide, dialyzed free 

 of unreacted reagent, and oxidized with performic acid as before. The oxi- 

 dized protein was then digested with trypsin and chromatographed with the 

 eluting system shown above. The chromatogram obtained resembled the 

 one shown in Fig. 4. Each fraction from the soluble portion of the digest 

 was hydrolyzed with acid and examined for the presence of carboxymethyl- 

 cysteinesulfone. None of these fractions, pure or impure, known to contain 

 cysteic acid contains this derivative. However, the insoluble fraction con- 

 tained both cysteic acid and the sulfone. These preliminary findings pro- 

 vide strong evidence that iodoacetamide does not react in a random manner 

 with all the sulfhydryl groups of papain. On the contrary, the results indi- 

 cate that only a single sulfhydryl group in the papain molecule reacts with 

 the alkylating agent. Attempts to isolate and identify the peptide contain- 

 ing the carboxymethylcysteinesulfone have, so far, not been successful. The 

 insoluble residue appears to contain several components and the limited 

 solubility of these peptides in acid solution presents a particularly difficult 

 problem of resolution. 



