Preparation and properties of water-insoluble derivatives of trypsin.

The conversion of enzymes into water-insoluble products possessing specific catalytic activity is of interest, since such water-insoluble enzyme derivatives can readily be removed from the reaction mixture and can be used for the preparation of columns with enzymatic activity. When stable, they can be employed repeatedly to induce specific chemical changes in relatively large amounts of substrate. Incubation of substrate with a water-insoluble enzyme for a short period may facilitate the detection and isolation of intermediates formed at the early stages of an enzymatic reaction. Finally, it might be expected that the study of the properties of water-insoluble enzymes might shed new light on the mode of action of enzymes present in cell membranes. Adsorption techniques have been applied to combine enzymes with insoluble carriers (2, 3). These procedures led, however, to partial denaturation and resulted in preparations from which the reversibly adsorbed enzyme was liberated while in contact with substrate. In order to obtain irreversible binding between enzyme and carrier, attachment by covalent bonds seems necessary. Such binding should obviously be carried out via functional groups not essential for enzymatic activity. Some antigens and antibodies have been attached by chemical bonds to water-insoluble high molecular weight compounds (4-8). The immunologically active columns of this kind were used in the isolation of both antibodies and antigens. Few papers have been published on the chemical binding of enzymes to water-insoluble polymers. The coupling of pepsin and carboxypeptidase with a diazotized poly-p-aminostyrene was reported by Grubhofer and Schleith (9). A low enzymatic activity was recorded for the water-insoluble enzymes obtained, which were poorly characterized. The synthesis of diazobenzyl cellulose and carboxymethyl cellulose derivatives of trypsin and chymotrypsin was described by Mitz and Summaria (10). Carboxymethyl cellulose azide was used in the synthesis of the carboxymethyl cellulose derivatives. The water-insoluble enzyme preparations were found to be more stable than the corresponding water-soluble enzymes. Epstein and Anfinsen (11) succeeded recently in reversibly reducing the disulfide bonds of trypsin bound to carboxymethyl cellulose. Free trypsin could not be used, as its reduction was accompanied by precipitation