ACTION OF IMMOBILIZED XANTHINE-OXIDASE ON PURINES

Xanthine oxidase was covalently immobilized on polyacrylamide gel bead s, polyamide-11 and dacron. Hypoxanthine (15 ml of 200 mu M), prepared in 0.1 M phosphate buffer, pH 8.0, was circulated through a column co ntaining 1.0 g derivatized enzyme at a flow rate of 1.0 ml/min at 28 d egrees C. Specific activites of 0.660, 0.072 and 0.016 Units/mg of pro tein were demonstrable for the polyacrylamide gel beads, dacron and po lyamide-11 derivatives, respectively. The action of these water insolu ble enzyme derivatives on 6-mercaptopurine (15 ml of 660 mu M) was als o investigated, under the same experimental conditions, showing specif ic activities of 0.063 Units/mg, 0.574 mu Units/mg and 0.118 mu Units/ mg, respectively. The 6-mercaptopurine oxidative pathway catalyzed by immobilized xanthine oxidase on dacron stopped at the intermediate com pound, 6-mercapto-8-hydroxypurine, so that no 6-thiouric acid was prod uced, whereas the immobilized preparations using polyacrylamide gel be ads and polyamide-11 behaved like the soluble enzyme, namely, 6-thiour ic acid was the final product. The behavior of dacron-xanthine oxidase compound was similar to that previously described for the derivatives obtained with carboxymethylcellulose and chitosan. The hypoxanthine o xidative pathway catalyzed by xanthine oxidase immobilized on these th ree supports was similar to the soluble enzyme. However, although its oxidation is stoichiometric for polyacrylamide gel beads and polyamide -11 derivatives, and no xanthine formation is observed (steady-state e quilibrium), under the action of the enzyme-dacron derivative the xant hine formation rate (0.164 mu Units/mg) is higher than the uric acid f ormation rate (0.017 mu Units/mg) compared to the hypoxanthine consump tion (0.072 mu Units/mg). These findings suggest again that xanthine o xidase-dacron derivative is limited to the catalysis of oxidation of h ypoxanthine carbon atom number 2 as in 6-mercaptopurine.