Bovine milk xanthine oxidase: effect of limited proteolysis on kinetics and structure

Bovine milk xanthine oxidase was purified by an improved nonproteolytic method. This enzyme and a proteolytically purified preparation were kinetically characterized and compared. Even though there were some similarities between the two preparations in Q sub(10) and energy of activation, proteolytically derived xanthine oxidase (PDXO) migrated faster in electrophoresis and its kinetic behavior was significantly different than that of nonproteolytically derived xanthine oxidase (NPDXO). At subsaturating substrate concentrations, the activity of PDXO was much less in 0.1 M Tris-HCl buffer than in 0.1 M pyrophosphate buffer but the activity of NPDXO was the same in both buffers. The data suggest that proteolysis, by modifying the primary structure, has reduced the enzyme's conformational stability, catalytic efficiency, and affinity for both substrates and competitive inhibitors.