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



Subsequent patterns (Fig. 4) have shown only traces of ninhydrin-positive 

 material in the regions where Fractions 1, 2, and 3 were expected and ana- 

 lyses of the small peaks in these areas have revealed only small amounts of 

 material with free cysteic acid predominating in the region of Fraction 1 

 and free aspartic acid in the region of Fraction 2.^° In later portions of the 

 chromatogram reproducibility has been much better. Fractions 5 through 16 

 have appeared constantly in their expected positions. The complexity of the 

 fractions is, however, quite variable. Fraction 4 (Fig. 3), for example, appears 

 to be a mixture of small amounts of a number of peptides or amino acids, 

 and the shape of the curve shown here is certainly suggestive of this. Frac- 

 tions 10 plus 1 1 (Fig. 4) are composed of at least three peptides, and Fractions 

 12 and 13 overlap with the same large complex peptide. Fractions 5, 6, 8 

 and 9 appear to be constant in character. 



■ I I i_ 



720 760 800 840 880 920 

 FRACTION NUMBER 



960 1000 



Fig. 4. Chromatographic separation of peptides in a tryptic digest of oxidized papain. 

 Except for the absence of Fractions 1 to 3, the results were similar to those shown in 

 Fig. 3. (From Kimmel and Smith, unpublished.) 



Table 4 shows the composition of the peptides that have been isolated 

 and shown to be pure.^" Unknown sequences are enclosed in parentheses. 

 It is interesting that two of the sequences shown have been encountered 

 in other proteins and polypeptides. The sequence serylarginine has been 

 found in chymotrypsin^^ and in ribonuclease.^^ The very unusual sequence 

 ala.ala.ala.lys. is also present in ribonuclease.^^ 



It is obvious that the peptides shown in Table 4 represent less than one 

 third of the papain molecule. The yield of these peptides is not high, ranging 

 from 20 to 40 per cent, but significant enough to indicate that they repre- 

 sent authentic fragments from oxidized papain. Several of the fractions 

 shown in Figs. 3 and 4 are known to be mixtures of peptides, and attempts 



