Purification of xanthine oxidase from the fat-globule membrane of bovine milk by electrofocusing.

Xanthine oxidase was purified from bovine milk-fat globule membrane by extraction with butan-1-ol, precipitation with ammonium sulphate, separation by preparative electrofocusing and chromatography on Concanavalin-A/Agarose. The enzyme had an A280/A450 ratio of 4.8 and a specific activity of 3.09. At least five to seven variants of the enzyme with isoelectric points from pH 6.9 to 7.6 were identified. Previously identified minor "variants' of the enzymes with apparently acidic isoelectric points (1) were shown to be the result of aggregation of enzyme with membrane sialoglycoproteins. Specific antibodies to xanthine oxidase were prepared by fractionating immune serum on a column of enzyme covalently bound to Sepharose 4B. A single immunoprecipitate was obtained when the purified antibodies were allowed to diffuse in agarose gels against either Triton-X-100-extracted membrane or purified xanthine oxidase. Immunoelectrophoresis of the enzyme against anti-sera to xanthine oxidase, however, revealed two precipitin lines, both of which were positive when histochemically stained for enzyme activity. The results are discussed with reference to previous purification schemes for xanthine oxidase and previous estimates for the isoelectric points of the enzyme. We also outline practical uses for the antibody prepared against the enzyme in this present study.