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



derivative has been accomplished by the following procedure.^ The reduced 

 enzyme was allowed to react with one equivalent of iodoacetamide. The 

 protein derivative was then exhaustively dialyzed and the preparation was 

 oxidized with performic acid. An aliquot was hydrolyzed with 6n-HC1 under 

 the usual conditions and worked up in the conventional manner. Chromato- 

 graphy of a sample of the hydrolysate showed the presence of carboxy- 

 methylcysteine sulfone which was identical in behavior with that of an 

 authentic, synthetic sample of this compound. Thus, there is no question 

 that the reactive thiol group is present on a residue of cysteine. 



The aforementioned studies with PCMB, iodoacetamide and with mercury^ 

 are in accord with the view that there is only one reactive thiol group in 

 the enzyme. However, it is essential to ascertain the total number of such 

 groups in the protein. Preparations of papain, treated by various methods, 

 react with more than one mole of PCMB. Under certain conditions, it has 

 been found that the enzyme will react with 6 moles of the mercurial, as 

 judged by mercury analyses,*-^ These results indicate that 6 thiol groups 

 are present in the molecule. Inasmuch as hydrolysates of oxidized papain 

 yield 6 moles of cysteic acid,^^ the agreement between these results suggests 

 that cystine itself, and hence that disulfide bridges, are absent from the 

 papain molecule. 



It has been reported^^'^^ that methionine is absent from papain. Never- 

 theless, elementary analysis reveals that the molecule contains 8 atoms of 

 sulfur^-^ and the presence of only 6 cysteine residues leaves two atoms of 

 sulfur to be explained. We have been able to exclude the presence of inor- 

 ganic sulfate, acid-labile sulfate esters, thiolhistidine, or other known amino 

 acids containing sulfur.^ Thompson^^ could not detect lanthionine in hydro- 

 lysates of papain. It is our hope that the present systematic investigation 

 of the structure will provide eventually an understanding of the nature of 

 the two missing sulfur atoms. 



STUDIES OF STRUCTURE 



Amino terminal groups. With the dinitrophenyl method of Sanger,^' Thomp- 

 son'^ demonstrated that mercuripapain contains only one free a-amino group, 

 that of isoleucine. From partial hydrolysates of the dinitrophenyl protein, 

 he isolated the unique tripeptide, DNP-isoleucylprolylglutamic acid. These 

 findings indicate that the papain molecule consists of only a single peptide 

 chain. In addition, Thompson isolated 8 moles of e-DNP-lysine which is 

 in accord with the number of lysine residues found by amino acid analysis.^* 



Peptides containing cysteic acid. Inasmuch as at least one cysteine residue is 

 of primary importance in the catalytic activity of papain, a study was made 

 of the strongly acidic peptides which could be isolated from partial acid 

 hydrolysates of papain which had been oxidized with performic acid.^^ 

 Hydrolysis of the oxidized protein was performed in 12N-HC1 at .37° for 



