Solubilization and purification of xanthine oxidase from bovine milk fat globule membrane

Bovine milk xanthine oxidase (XO) was isolated and purified from milk fat globule membrane (MFGM). The method included the following steps: solubilization of XO from MFGM in 200 mM dithiothreitol (DTT) at pH 8.0, fractionation of solubilized proteins with ammonium sulfate, chromatography on DEAE-Sepharose with gradient elution, and rechromatography of the XO fraction for final purification. The method is highly reproducible, is comparatively simple, and provides highly pure enzyme. Purified XO, analyzed by (8%) SDS-PAGE, had only one band of 140-150 kDa. XO showed a high specific activity of 2.5 units/mg of protein and an A280: A450 ratio of 4.8.