Reaction with iodoacetamide and the number of active centres in xanthine oxidase

Abstract

Active milk xanthine oxidase has 2 moles of FAD, 2g.atoms of Mo and 8g.atoms of Fe/mole of protein of mol.wt. 275000 (Hart, McGartoll, Chapman & Bray, 1969). There has been a tendency to assume (cf. Massey, Brumby, Komai & Palmer, 1969) that all of these constituents would not be likely to function as a single catalytic unit in the molecule and that there must be two presumably equivalent active centres (Bray, 1963). We now present evidence based on FAD loss (Bray, Chisholm, Hart, Meriwether & Watts, 1966) during inactivation of the reduced enzyme by iodoacetamide (Bray & Watts, 1965) indicating that xanthine oxidase operates, during aerobic xanthine oxidation at least, with a single active centre.The interpretation of earlier quantitative studies on reaction ofxanthine oxidase with iodoacetamide, by measuring iodide liberation (Bray & Watts, 1965) and following FAD loss (A. J. Chisholm, A. M. Scott & R. C. Bray, unpublished work), was complicated by the use as starting material of preparations with non-stoicheiometric FAD/Mo ratios. These problems were overcome for the present work by the use of the salicylate method for preparing the enzyme, this giving samples free from demolybdoxanthine oxidase (Hart et al. 1969). The enzyme used was also free from detectable impurities in the ultracentrifuge and in recycling gel filtration on Sephadex G-200. Techniques and analytical methods were as described by Hart et al. (1969). Reaction of enzyme, reduced with xanthine, with iodo[14C]acetamide was carried out at pH5-5 to minimize side reactions (Bray & Watts, 1965) and was terminated by the addition of cysteine. The product from the reaction was dialysed, then purified by recycling gel filtration on Sephadex G-200. After three cycles it showed a single symmetrical peak, radioactivity (detected with a flow scintillation counter from Packard Instruments Ltd., Wembley, Middx.), residual xanthine oxidase

Noncompetitive and irreversible inhibition of xanthine oxidase by benzimidazole analogs acting at the functional flavin adenine dinucleotide cofactor

“…The above findings bear a close resemblance to those of , McGartoll and Bray (1969), and Massey (1969, 1971) regarding iodoacetamide inactivation of the enzyme. The latter workers showed that formation of a C(4a) adduct of the reduced functional FAD is the primary process in inactivation.…”