NADH oxidation and superoxide production by caprine milk xanthine oxidoreductase

Xanthine oxidoreductase (XOR) was purified from caprine milk where more than 90% exists in demolybdo 'inactive' form. The dehydrogenase form of the enzyme, oxidises nicotinamide adenine dinucleotide (reduced) (NADH), in the presence of oxygen, and generates superoxide anion radical (O2−) significantly faster than does the oxidase form. The corresponding forms of human and bovine milk enzymes behaved similarly. The steady-state kinetics of NADH oxidation and O2− production, in the absence and presence of NAD+, by the dehydrogenase form of XOR from the three species, are analysed. Allopurinol, oxypurinol and amflutizole blocked the reducing substrates that act at the molybdenum site, and all of which were ineffective in the case of NADH oxidase activity. However, and as expected, diphenyleneiodonium was a powerful inhibitor of NADH oxidation. The possible physiological and pathological significance of reactive oxygen species, especially O2−, arising from NADH oxidation by caprine XOR is discussed.