Reaction of milk xanthine oxidase with o-phenanthroline and 1,7-dimethylxanthine: a mechanistic study

The kinetics of the reaction of milk xanthine oxidase (XO) with o-phenanthr oline and 1,7-dimethylxanthine are investigated by optical and circular dic hroism (CD) spectroscopic techniques. o-Phenanthroline and 1,7-dimethylxant hine are observed to react with XO, resulting in a slow decrease in the abs orbance and CD signals of the enzyme. The results suggest the existence of a multi-binding mechanism in the reaction of XO with o-phenanthroline with two molecules of the substrate binding to one molecule of the enzyme. Howev er, 1,7-dimethylxanthine binds reversibly with the enzyme with 1 : 1 stoich iometry. The kinetic effect experienced individually by the flavin adenine dinucleotide (FAD) chromophore of XO has been separately resolved, and show s that at least one molecule of o-phenanthroline may bind near the FAD cent re of the enzyme. 1,7-Dimethylxanthine is expected to bind to the molybdenu m centre in analogy with xanthine.