The chemistry of xanthine oxidase. Reaction with iodoacetamide

1. The reaction of milk xanthine oxidase with iodoacetamide has been studied with the silver-silver iodide electrode. 2. The reaction proceeds considerably faster in the presence of xanthine than in its absence. Anaerobically, with excess of xanthine, the reaction takes place as a rapid phase in which the enzyme is inactivated and in which approx. 1 thiol group/mol. of enzyme reacts and as a slower phase in which about 12 groups/mol. react. 3. The rapid reaction appears to be first-order with respect to xanthine oxidase and iodoacetamide and independent of the xanthine concentration with more than about 3mol. of xanthine/mol. of enzyme. 4. The velocity constant of the rapid phase is 0.26min.(-1) at 25 degrees and pH7.0, with 1mm-iodoacetamide and 17mum-xanthine oxidase. The velocity constant for the slower phase is about one-hundredth of this value. 5. The velocities of both phases increase with increasing pH in the range 5.0-9.6. 6. Xanthine may be replaced by salicylaldehyde without affecting the rate of loss of enzymic activity. With sodium dithionite as reducing agent, the reaction is slightly faster. 7. The possible function of thiol groups in the reaction mechanism of the enzyme is discussed.