Phenolic hydroxyl ionization in papain.

Various properties of crystalline papain have been under investigation in this laboratory for several years in attempts to understand the specificity, mode of action, and other aspects of the behavior of this enzyme (1). Inasmuch as papain must be in its native state to retain its enzymic activity, a knowledge of the role of the amino acid side chains in maintaining conformation is essential. The difference spectrum obtained by comparison of papain in acid and neutral solution exhibits peaks at 279 and 287 rnp (2) and is suggestive of the involvement of the phenolic hydroxyl groups in hydrophobic or hydrogen bonding. Crammer and Neuberger (3) developed a spectrophotometric procedure to investigate the ionization of phenolic groups in ovalbumin and insulin. Similar investigations have since been performed on poly-L-tyrosine (4), serum albumin (5), ribonuclease (6,7), lysozyme (8,9), chymotrypsinogen (lo), trypsinogen (111, myosin, and the meromyosins (12). These studies have shown marked differences in the ionization behavior of phenolic groups in various proteins. This paper presents an investigation of the behavior of the phenolic hydroxyl groups in papain by means of spectrophotometric titration of this protein in the range from pH 5.5 to 14.