Preparation of high purity xanthine oxidase from bovine milk

Bovine milk xanthine oxidase, referred to herein as XO, is isolated and purified from raw whole milk by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Sodium salicylate, ethylenediaminetetraacetate (EDTA), and a 0.2 M phosphate buffer are added to fresh milk. After incubation at 40 DEG -45 DEG C. for 105 min., the mixture is adjusted to 1-2% with Triton X-100 and allowed to incubate for 15 min. The mixture is cooled to 4 DEG C., followed by a 2-step fractionation of the proteins with ammonium sulfate. The crude enzyme is isolated as a red-brown precipitate which is dissolved in 0.1 M Tris/CaCL2 buffer and stored for from 12 to 168 hours at -20 DEG C. The isolated enzyme is purified by column chromatography (Sephadex G-75, Sephacryl S-200, Sepharose 6B, and Sephadex G-75). The final stage of purification is accomplished by passing the enzyme preparation through a DEAE-Sephadex A-50 column in a continuous linear salt gradient from 0.005 M to 0.1 M pyrophosphate buffer.