II] STRUCTURE AND ACTIVITY OF PAPAIN 189 



column 7 mm. in diameter and represents the first successful attempt to 

 resolve the tryptic digest of oxidized papain. Subsequent chromatography 

 was performed on a column 30 mm. in diameter having 17 times the capacity 

 of the original column. In general, the transition from the small to the large 

 column has resulted in only minor changes in the elution pattern. ^^ 



5PH 

 O 3 

 z 2 



g A 



>- 



X 



^ 2 



I 



-I' V2 



2 



-< >- < — 



1 1 !_- ^J ^l L^ ^J I I I L_ ; 



20 60 140 170 200 300 340 380 4 20 460 500 



540 580 620 



780 820 860 

 FRACTION NUMBER 



900 



1380 1420 



Fig. 3. Chromatographic separation of soluble peptides found in a tryptic digest of 

 oxidized papain. The ninhydrin-reactive components were resolved on a 150 cm. column 

 of Dowex-50x2 with buffers varying in ionic strength and pH. The solid bars show the 

 fractions which were pooled. (Kimmel and Smith, unpublished.) O.D. is optical density; 

 upper part of figure : full lines, pH ; broken lines, ionic strength. 



Amino acid analyses have been performed on all of the fractions numbered 

 in this figure. Most of the fractions up to Fraction 14 appear to contain 

 single peptides or mixtures of peptides in which each peptide contains only 

 one basic amino acid. Fractions 14 through 18 contain mixtures of large 

 complex peptides with more than one basic amino acid. Presumably, these 

 result from incomplete tryptic digestion, although it is possible that the 

 peptide bonds involving the linked carboxyl groups of lysine and arginine 

 in these fragments are resistant to trypsin, as in a lysylproline sequence. 



Fractions 1, 2 and 3 in this chromatogram emerge as sharp symmetrical 

 peaks and the stoichiometry of the amino acid analyses suggested that these 

 fractions were composed of nearly pure peptides. Fraction 1 is particularly 

 interesting since analysis showed 27 amino acid residues including 2 resi- 

 dues of cysteic acid. No basic amino acid was found in this fraction and, 

 therefore, it was assumed that fraction 1 represents the C-terminal portion 

 of papain. ^^ Fraction 2 is a smaller peptide with a high aspartic acid content. 



The elution pattern shown in Fig. 3 was obtained on 5 different occasions. 



