Purification and characterization of xanthine dehydrogenase from Clostridium acidiurici grown in the presence of selenium

Abstract

Xanthine dehydrogenase has been purified from Clostridium acidiurici to homogeneity. The purified enzyme exhibited a specific activity of 385 units per mg protein with xanthine as substrate and methyl viologen as most effective electron acceptor. However, the enzyme activity was quite unstable. Michaelis constants for xanthine and methyl viologen were 1.35 and 0.44 mM, respectively. Uric acid, xanthine, hypoxanthine, 6-mercaptopurine, allopurinol and purine could serve as substrates. NAD, NADP and ferredoxin were inactive as acceptors. The molecular mass of the enzyme was estimated to be 224 kDa. After SDS-gel electrophoresis, five different polypeptides were detected. Analysis of the native enzyme gave values for non-haem iron, acid-labile sulphur, FAD, molybdenum, tungsten and selenium in a molar ratio of 7.7:7.5:1.7:1.8:0.12:0.13, respectively. Free ESR radical signals, presumably due to falvin semiquinone, were observed after reduction with xanthine, dithionite, or NADH. Despite the requirement for selenium for the synthesis of the active xanthine dehydrogenase, there was no evidence for involvement of selenium in the iron-sulphur or molybdenum centres. Reduction with xanthine induced a signal analogous to the rapid Mo(V) species of other xanthine dehydrogenases and xanthine oxidase, indicating that active molybdenum centres are present. In addition, a ‘slow’ desulphomolybdenum signal was observed after prolonged reduction with dithionite, and a ‘desulpho-inhibited’ signal in the protein reduced with dithionite plus 9 M ethanediol. Spectra of two iron-sulphur centres I + II, corresponding to the [2Fe-2S] clusters of milk xanthine oxidase, were observed after reduction with dithionite.