Kinetic Analyses of Two-Steps Enzymatic Oxidation from Hypoxanthine to Uric Acid with Xanthine Oxidase by Capillary Electrophoresis/Dynamic Frontal Analysis

Abstract

Two steps of enzymatic oxidations from hypoxanthine to uric acid with xanthine oxidase (XOD) were kinetically analyzed by capillary electrophoresis/dynamic frontal analysis. When a substrate solution of hypoxanthine was introduced into a capillary with a separation buffer containing XOD, the enzymatic reaction continuously proceeded during the electrophoresis and a product of xanthine was continuously resolved from the substrate zone. A plateau signal of the product xanthine was detected based on the constant reaction rate with XOD. The plateau height was directly related with the reaction rate, and a MichaelisMenten constant K_M,HXA was successfully determined as 770±40 μmol L⁻¹. When xanthine was used as a substrate, a slope response of uric acid was obtained because of the low concentrations of the substrate and its significant decrease. However, the Michaelis-Menten constant was successfully determined by using the initial reaction rate, and a Michaelis-Menten constant of K_M,XA was determined as 85±6 μmol L⁻¹.