Preparation and characterization of polytyrosyl trypsin.

The preparation of polypeptidyl proteins was first reported by Becker and Stahmann (l), who showed that the polymerization of N-carboxyamino acid anhydrides may be initiated by the free amino groups of proteins to yield the corresponding protein derivatives. Becker, Stahmann, et al., as well as a number of other investigators, have since reported on the physicochemical and biological properties of a large number of such derivatives (2-12). Generally, it has been found that the polypeptidyl derivatives obtained under the mild conditions employed are reasonably homogeneous as judged by sedimentation, electrophoresis, and immunoelectrophoresis (3-6). Furthermore, little change in the configuration of the modified proteins has been noticed. Chemical analyses suggest that the polypeptidyl side chains are attached to the (Yand c-amino groups of the modified proteins (7, 8, 11). Polypeptidyl derivatives of chymotrypsin and ribonuclease retained full enzymatic activity (10, 11). The preparation in this laboratory of a water-insoluble trypsin via a polytyrosyl trypsin has recently been described (13). The water-insoluble enzyme was prepared by coupling polytyrosyl trypsin with a diazotized copolymer of p-amino-DL-phenylalanine and L-leucine. It showed towards benzoyl-L-arginine methyl ester approximately one-fifth of the esteratic activity of trypsin, possessed a high resistance toward autodigestion, and could only be partially inhibited by soybean and pancreatic trypsin inhibitors (14). The work presented in this paper was carried out to determine the physical, chemical, and enzymatic properties of polytyrosyl trypsin in order to determine whether some of the differences in the properties of crystalline trypsin and water-insoluble trypsin could be attributed to the tyrosination. The preparation and characterization of various polytyrosyl proteins is desirable, since such derivatives are most suitable for coupling, by means of diazo links, to water-insoluble carriers. Polytyrosyl chymotrypsin and polytyrosyl ribonuclease were used in a manner similar to polytyrosyl trypsin as intermediates in the preparation of the corresponding water-insoluble enzymes.’ Polytyrosyl trypsin was prepared by initiating the polymerization of N-carboxy-L-tyrosine anhydride (15) in aqueous solution with trypsin. A similar procedure was previously used for the preparation of polytyrosyl gelatin (12). Polytyrosyl trypsin