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



on ion-exchange columns by the procedures of Moore and Stein. ^® In our 

 studies, ^^ we used different times of hydrolysis in 6n-HC1 in order to cor- 

 rect for destruction of labile amino acids and to produce complete libera- 

 tion of slowly released residues. Tryptophan was estimated by a colorimetric 

 method^^ and cysteine was estimated chromatographically after hydrolysis 

 of the oxidized protein. ^^ 



The composition of papain shows several noteworthy features: the ab- 

 sence of methionine, the presence of only four residues of phenylalanine 

 and one of histidine, and the high content of glycine and tyrosine. 



Tryptic digestion of oxidized papain. The most useful approach to the study 

 of amino acid sequence in large polypeptides and proteins has been to de- 

 grade the molecule first into a relatively small number of large peptides. 

 Experienced'-^ has shown that this type of degradation can best be accom- 

 plished with proteolytic enzymes. Selection of the proteolytic enzyme de- 

 pends upon the nature of the protein being studied and the specificity of 

 the enzyme. With papain, trypsin appeared to be the enzyme of choice for 

 first study. Papain contains 9 arginine and 8 lysine residues, thus providing 

 17 sites at which trypsin can act and this would yield, theoretically, 18 pep- 

 tides. The amino acid composition of papain is such that other proteolytic 

 enzymes could be expected to yield a larger number of fragments. 



Papain itself, mercuripapain, and papain combined with 6 moles of PCMB 

 are relatively resistant to the action of trypsin. ^^ Furthermore, each of these 

 materials is potentially capable of being partially activated during tryptic 

 digestion, thus permitting autolysis and inactivation of the trypsin. Auto- 

 lysis is particularly undesirable because of the broad specificity of papain. 

 The most satisfactory papain derivative for degradation by trypsin is the 

 performic acid-oxidized protein. This derivative has no proteolytic activity 

 and is digested by trypsin at pH 7-5. However, oxidized papain is insoluble 

 at this pH and digestion proceeds slowly for several days. Oxidation of 

 papain results in modification of the 5 tryptophan residues in the protein 

 as well as in oxidation of cysteine to cysteic acid residues.^^ 



The procedure has been to digest oxidized papain at pH 7-5 with crystal- 

 line trypsin until the ninhydrin color of an aliquot is constant. In general, 

 fresh trypsin has been added every 10 to 20 hours. In no case did the trypsin 

 content of the digestion mixture exceed 2 per cent of that of the oxidized 

 papain. The resulting digest is dried from the frozen state and the dry 

 material is extracted with a 0-2 n formate buffer at pH 2-1. The soluble 

 peptides in the extract are resolved by chromatography. The large amount 

 of insoluble residue must be fractionated by other means. 



Chromatography of the soluble peptides has been accomplished on a 

 150 cm. column of Dowex 50 x 2 with a gradient elution system. The elution 

 pattern and the pH and ionic strength gradients are shown in Fig. 3.^^ It 

 should be noted that this particular chromatogram was obtained on a 



