A two-step procedure for purification of papain from extract of papaya latex.

A method is presented for purifying papain from extracts of papaya latex. The procedure involves precipitation of the extract of papaya latex with sodium chloride followed by affinity chromatography of the redissolved precipitate. Precipitation of the protein from the latex extract is necessary to separate the papain from material which interferes with the binding of papain to the affinity column. During affinity chromatography, the affinity column is overloaded to insure absence in the final product of impurities which are capable of binding to the affinity column. The papain prepared by this procedure yielded an amino acid analysis and an N-terminal amino acid analysis expected for a sample of pure papain. No Met was detected on amino acid analysis nor was the presence of N-terminal residues other than Ile detected. On polyacrylamide disc gel electrophoresis at pH 4.3, papain prepared by the method described in this work was indistinguishable from crystalline papain which was prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Both disc gel patterns consisted of a single band and a trailing shadow which was less than 5% of the main band. In routine spectrophotometric assays, the specific activity toward N, o-benzoyl-L-arginine ethyl ester of papain prepared by the procedure described in this work was indistinguishable from crystalline papain prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Values of 24 set ’ and 15 mM were obtained from the turnover number and K, for the papain-catalyzed hydrolysis of N, a-benzoyl-L-arginine ethyl ester at 25”C, pH 6.00, I/2 0.30 using a pH stat.