Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose

The oxidase form of xanthine dehydrogenase (XO; EC 1.1.3.22) has been purified approximately 200-fold from rat liver extracts using a three-step process of heat treatment, ammonium sulfate precipitation, and chromatography on benzamidine-Sepharose. The purified enzyme showed only minor contamination when analyzed by gel electrophoresis under either native or sodium dodecyl sulfate (SDS)-denatured conditions and appears to be intact based on its subunit size on SDS-polyacrylamide gel electrophoresis, its N-terminal amino acid sequence, and its ability to be converted to the NAD-dependent dehydrogenase form (XD; EC 1.1.1.204) by incubation with dithiothreitol, Isoelectric focusing analysis showed that the purified enzyme consists of two major, enzymatically active isoforms with average pI values of 6.13 and 6.23 and a minor enzymatically active isoform with an average pI value of 6.07, A similar purification of XD was achieved by preincubating the partially purified oxidase with dithiothreitol prior to affinity chromatography on benzamidine-Sepharose. The effects of benzamidine on the kinetic properties of purified rat XO were characterized at pH 8 and 9 and were compared to those of bovine milk XO, Benzamidine was found to be a weak competitive inhibitor of the purified rat enzyme with K-i values of 30 and 10 mM at pH 8 and 9, respectively, In contrast, the K-i values for benzamidine with bovine XO were more than 10-fold greater. The findings presented in this study show that benzamidine is a competitive inhibitor of XO and that affinity chromatography on benzamidine-Sepharose provides a simple, rapid, and effective means of purifying both the oxidase and dehydrogenase forms of rat XO. (C) 1996 Academic Press, Inc.