Simple, high-yield purification of xanthine oxidase from bovine milk

Xanthine oxidase, a commercially important enzyme with a wide area of appli cation, was extracted from fresh milk, without added preservatives, using t oluene and heat. The short purification procedure, with high yield, consist ed of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast how) column chromatography. Xanthine oxidase was eluted as a single activit y peak from the column using a buffer gradient. The purification fold, spec ific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in s pecific activity was observed. Activity and protein gave coincident stainin g bands on native polyacrylamide gels. The intensity and the number of band s were dependent on the oxidative state(s) of the enzyme; reduction by 2-me rcaptoethanol decreased the intensity of the slow-moving bands and increase d the intensity of the fastest-moving band. Following sodium dodecyl sulfat e-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for greater than or e qual to 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purif ied xanthine oxidase becomes a heterodimer due to endogenous proteases. (C) 1999 Elsevier Science B.V. All rights reserved.