title: The interaction of bisulfite with milk xanthine oxidase.
abstract: Bisulfite ion competitively inhibits xanthine oxidase activity. The ability of HSO3- to bind at the molybdenum center is controlled by pH due to a pKa of 6.91 for SO3(2-)/HSO3-. The Kd for the enzyme-bisulfite complex is 4.5 x 10(-5) M at pH 7.0 and 25 degrees C. The relative magnitude of extinction changes in the optical absorption spectra, the number of inhibitor ions reversibly bound, and the number of electrons required for complete bleaching of the visible spectrum of the milk xanthine oxidase-HSO3- complex were all dependent on the percentage of fully functional xanthine oxidase. Binding of HSO3- causes perturbations of the visible spectrum: the maximum extinction changes at 320 and 422 nm were calculated to be -4300 and -2150 M-1 cm-1, respectively. The stoichiometry of reversible binding was determined to be one molecule of HSO3-/active molybdenum center. Combined optical and EPR analyses of anaerobic dithionite titrations revealed that the relative redox potentials of the Mo6+/5+ and Mo5+/4+ couples decreased by approximately 35 and 45 mV on binding bisulfite, respectively. The finding that bisulfite has a profound effect on the redox properties of xanthine oxidase necessitates a re-evaluation of dithionite titrations previously carried out with this enzyme at neutral and low pH values since bisulfite produced as an oxidation product of dithionite binds to the enzyme during the course of titration.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
authors.forename: K M
authors.initials: KM
authors.lastname: Fish
authors.forename: V
authors.initials: V
authors.lastname: Massey
authors.forename: R H
authors.initials: RH
authors.lastname: Sands
authors.forename: W R
authors.initials: WR
authors.lastname: Dunham
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008892:Milk / Q000201:enzymology*; D008982:Molybdenum / Q000378:metabolism; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013447:Sulfites / Q000378:metabolism / Q000494:pharmacology*; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D013447:Sulfites; D008982:Molybdenum; D014969:Xanthine Oxidase; C009279:sodium bisulfite