The nature of catalytic activities of milk xanthine oxidase

Abstract

Xanthine or hypoxanthine anaerobically reduced flavin-adenine dinucleotide (FAD) of all xanthine oxidase prepns. studied in 2 ways: (a) a rapid reduction, occurring within 30 sec; (b) a slow reduction up to 60 min. or more. The activity per unit total FAD of 4 enzyme prepns. was proportional to the percentage of FAD rapidly reducible by xanthine. This confirmed the generally held belief that the FAD of active xanthine oxidase acts as a H carrier. The slow reaction was due to the reduction of FAD attached to inactivated apo-enzyme by reduced FAD attached to active apo-enzyme. Xanthine oxidase, D-amino acid oxidase and notatin, in the presence of their substrates, anaerobically reduced free FAD so slowly that free FAD could not act significantly in vivo as a carrier linking these systems with other flavine dehydrogenases. CaCl2, MgSO4 and some other bivalent metal salts detached the FAD of xanthine oxidase from the apo-protein without precipitating the latter. Spectrophotometric analysis of this reaction showed that the linkage between FAD and the apo-enzyme increased the absorption due to FAD by a factor of 1.71 at 450 m[mu]. It was also responsible for the reddish color of xanthine oxidase prepns. There was no evidence for an additional colored component of the prosthetic group of this enzyme, first postulated by Corran et al. (ibid. 33:1694. 1939). The same FAD molecules in a xanthine oxidase prepn. acted as H carriers in the oxidation of xanthine or hypoxanthine, salicylaldehyde and xanthopterin. This was further very strong evidence that the enzymes xanthine oxidase, aldehyde oxidase and pterin oxidase, occurring in milk, are one and the same enzyme. The same molecules in xanthine oxidase prepn. did not act as H carriers in the oxidation of xanthine and reduced cozymase. Since other workers have shown that the active centers for these reactions are different, these enzymes must now be regarded as separate entities. The minimum value for the turnover number of xanthine oxidase was 313 min.-l at 19[degree] Xanthine oxidase had virtually no fluorescence when irradiated with blue light, compared to free FAD.