Aug 1, 1996

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

Archives of Biochemistry and Biophysics
J L McManamanJ E Repine

Abstract

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 pl 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 ...Continue Reading

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Mentioned in this Paper

Chromatography
Oxidase
Bos taurus
Dithiothreitol
Xanthine Oxidase
Benzamidine hydrochloride
Xdh
Aldehyde Oxidoreductases
Purification Aspects
Amino-Terminal Amino Acid

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