Chemical and Enzymic Studies on the Amino-terminal Sequence of Papain. a Reinvestigation.

Peptides derived from chymotryptic and tryptic digests of papain are under investigation, and the sequences of a number of these peptides have been reported earlier (1). The recognition of the NHz-terminal peptide of the protein from each digest is based on a knowledge of the NHz-terminal sequence of the intact protein. Thompson (2) established the first 3 residues as Ileu-Pro-Glu from his studies on dinitrophenyl papain. The chymotryptic peptide which is in agreement with this partial sequence is Ileu-Pro-Glu-Tyr-Val-Asp-Trp (3), and the tryptic peptide is Ileu-Pro-Glu-Tyr-Val-Asp-Trp-Arg (4). These peptides are identical for the first 7 residues, and are consistent with the NH2 terminus of papain. The sequence of 7 residues from the Edman degradation of papain was reported by Light and Smith (5) as Ileu-Pro-GluNHz-Ser-AspNHz-Val-Ser. Clearly, this sequence is not in accord with the sequences of the chymotryptic and tryptic peptides. In order to resolve this problem, the NHz-terminal sequence of papain has been reinvestigated. A modification of the paper strip method of the Edman procedure was applied to denatured papain. Each phenylthiohydantoin derivative was identified as well as the free amino acid regenerated from the derivative. The first 5 residues were established as Ileu-Pro-Glu-Tyr-Val. Attempts were also made to study the NHz-terminal sequence by enzymic methods. Although Hill and Smith (6, 7) reported that mercuripapain was hydrolyzed extensively by leucine aminopeptidase, a reinvestigation with highly purified leucine aminopeptidase indicated that mercuripapain is not significantly degraded by the enzyme. Evidence is presented that some preparations of the aminopeptidase are contaminated with an endopeptidase which can attack native mercuripapain.