Xanthine oxidase. III. Sulfite oxidation as an ultra sensitive assay.

Previous papers in this series have reported the hypoxanthinedependent oxidation of sulfite by cyanide-inhibited xanthine oxidase (1) and experiments which permitted postulation of the structure of the active site of this enzyme (2). In the present study, xanthine oxidase acting on its substrates has been found to initiate the aerobic oxidation of sulfite by free radical chains of great length (3). Since each initiating event causes the oxidation of many sulfite ions, the latter process serves as an “amplifier” of the aerobic oxidation of purine substrates when followed manometrically, thereby permitting observation of the oxidation of purines at very low enzyme and substrate conccntrations. The various published Michaelis constants for the oxidation of hypoxanthine and of xanthinc by milk xanthine oxidase are in poor agreement. Application of the sulfite oxidation assay has permitted direct determination of these constants.