Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine

Abstract

9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthinelxanthine ox- idase reaction is important for 1) interpreting experi- mental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogen- esis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xan- thine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5- dimethyl- I-pyrroline-N-oxide or a combination of a- phenyl-N-tert-butyl-n&one and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl rad- ical formation was noted. Xanthine oxidase prepara- tions that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of HzOz, in the absence of xan- thine, to “high iron” xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or over- night dialysis of the enzyme against deferoxamine de- creased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxi- dase. However, commercial xanthine oxidase prepa- rations may contain adventitious iron bound to the