Determination of xanthine oxidoreductase forms: influence of reaction conditions

Xanthine oxidoreductase (XOR) has been implicated in ischaemia-reperfusion injury, and increases in this enzyme have been found in plasma of patients with different illnesses. The catalytic concentrations of the XOR forms found in plasma, using various reaction conditions, greatly differ in the related literature. We studied the effect of the assay conditions on the xanthine oxidation rate catalysed by the XOR forms. Our results demonstrate inhibition of XOR by the reaction products and a time-dependent decrease in the reaction rates of XOR forms. Substrate consumption and inhibition by the products did not account for this decrease. Determination at 60 min incubation leads to catalytic concentrations up to 80% lower for the XOR forms than those obtained at 10 min. We conclude that elimination of the reaction products (NADH, H(2)O(2) and O(2)) from the reaction mixture, and short incubation times, are necessary for accurate measurement of the XOR activities.