Determination of Kinetic Parameters for Immobilized Xanthine Oxidase and Its Application to a Post-Column Enzyme Reactor in High-Performance Liquid Chromatographic Analysis of Hypoxanthine, Xanthine and Uric Acid

Immobilized xanthine oxidase (XO) was prepared by intermolecular cross-linking to controlled-pore-glass (CPG) using glutaraldehyde, and the enzymic properties (pH optimum, stability change on immobilization and apparent Michaelis constant) were studied. The performances of enzyme reactors packed with the immobilized XO were considered theoretically and investigated experimentally. Using a post-column reactor packed with the immobilized enzyme, simultaneous determinations of hypoxanthine, xanthine and uric acid by high-performance liquid chromatography (HPLC) were achieved with detection at 290 nm. The coefficients of variation after 4 runs were 1.7-13.4, 0.3-9.0 and 3.7-31.4% in the determination of 0.405-1.620 μg/ml of hypoxanthine, 0.450-1.799 μg/ml of xanthine and 0.495-1.980 μg/ml of uric acid, respectively.