The chemistry of xanthine oxidase. Reaction with iodoacetamide

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Abstract
The reaction of milk xanthine oxidase with iodoacetamide has been studied with the Ag AgI electrode. 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 approximately 1 thiol group/mol. of enzyme reacts and as a slower phase in which about 12 groups/mol. react. The rapid reaction appears to be first-order with respect to xanthine oxidase and iodacetamide and independent of the xanthine concentration with more than about 3 mol. of xanthine/ mol. of enzyme. The velocity constant of the rapid phase is 0.26 min.-1 at 25[degree] and pH 7.0, with 1 m[image]-iodoacetamide and 17 [mu][image]-xanthine oxidase. The velocity constant for the slower phase is about 1/100 of this value. The velocities of both phases increase with increasing pH in the range 5. 0-9.6. 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. The possible function of thiol groups in the reaction mechanism of the enzyme is discussed.