Preparation and Properties of Water-insoluble Derivatives of Urease.

Water-insoluble enzyme derivatives possessing specific catalytic activity may provide a useful tool in biochemical studies, since they can readily be removed from the reaction mixture and can be used for the preparation of columns with enzymatic activity. The conversion of several enzymes into the corresponding water-insoluble derivatives by their binding via covalent bonds to insoluble carriers has been recently reported. The coupling of pepsin and carboxypeptidase with a diazotized polyp-aminostyrene was described by Grubhofer and Schleith (1). A low enzymatic activity was recorded for the water-insoluble enzymes obtained. The synthesis of azobenzyl cellulose and carbosymethyl cellulose derivatives of trypsin and chymotrypsin was reported by Mitz and Summaria (2). Carbosymethyl cellulose azide was used in the synthesis of the carbosymethyl cellulose derivatives. The water-insoluble enzyme preparations were found to be more stable than the corresponding watersoluble enzymes. Epstein and Bnfinsen (3) used a preparation of trypsin bound to carbosymethyl cellulose in their experiments on the reversible reduction of the disulfide bonds of this enzyme. Manecke (4) reported the synthesis of enzyme resins by allowing pepsin, diastase, or /3-fructosidase to react with a nitrated copolymer of methacrylic acid and methacrylic acid m-fluoroanilide. In our laboratory, water-insoluble derivatives of papain, trypsin, and chymotrypsin were prepared by using a diazotized copolymer of p-aminophenylalanine and leucine as insoluble carrier (5, 6). The water-insoluble papain derivative, possessing high proteolytic activity, was obtained by direct coupling of papain with the diazotized copolymer (7). It was used in the partial digestion of y-globulin, antibodies, and other proteins (7, 8). The water-insoluble trypsin (9, 10) was obtained by coupling polytyrosyl trypsin (11) with the diazotized copolymer of leucine and p-aminophenylalanine. It was more stable than native trypsin in the alkaline pH region, and was used in the preparation of a trypsin column with high and enduring tryptic activity. In this paper the preparation, properties, and some applications of water-insoluble urease derivatives are described. The most active and stable water-insoluble urease derivatives were obtained by binding urease, previously treated with p-chloromercuribenzoate, with the diazotized copolymer of p-amino-nnphenylalanine and L-leucine. A urease column was prepared and used in the assay of urea in body fluids as well as for the quantitative removal of urea from aqueous solution.