Preparation of bovine milk xanthine oxidase as a dehydrogenase form

Abstract

When xanthine oxidase was prepared from fresh raw cow's milk in the presence of dithioerythritol, 94 % of its xanthine-oxidizing activity was found as a dehydro-genase type. The enzyme was reversibly converted to an oxidase type when dithio-erythritol was removed. The conversion was ascribable to the oxidation of sulf-hydryl groups of the enzyme by oxygen. The two forms of the enzyme gave the same visible spectrum, but the dehydrogenase form alone gave a characteristic difference spectrum upon addition of NAD+. NADH served as a good electron donor for the dehydrogenase form of the enzyme but not for the oxidase form. When xanthine was used as an electron donor, the overall rate of />-benzoquinone reduction was the same for the oxidase and dehydrogenase forms, but the propor-tion of one-electron flux from the enzyme to /vbenzoquinone was considerably greater in the reaction of the dehydrogenase form than in that of the oxidase form. Xanthine oxidizing enzymes from mammalian sources were initially characterized as oxidases. In 1969, Stirpe and Delia Corte (1) reported tha