Purification of xanthine dehydrogenase from rat liver: a rapid procedure with high enzyme yields.

Arch Biochem Biophys. 1987 Oct; 258(1):219-25.AB

Xanthine dehydrogenase (EC 1.2.1.37) was purified approximately 1000-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (greater than 20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis.