Kinetics of Hydrogen Transfer between Hypoxanthine and Dimethylbiacridylium Nitrate in the Presence of Chick Liver Xanthine Dehydrogenase.

The chemiluminescence induced by the action of xanthine oxidase on its substrates in the presence of dimethylbiacridylium ions or 5-amino-a, 3-phthalae-l , 4-dione has been investigated by Totter et al. (l-5) and by Greenlee, Fridovich, and Handler (6). Tentative conclusions reached in the course of these studies were in agreement that the luminescence is probably dependent upon the formation of radicals and, in the case of lO,lO’-dimethyl9,9’-biacridylium ion, a suitable reduction product of the chemiluminescent substrate. It had been assumed by the former authors that the radicals formed were probably the result of the release of a stoichiometrically small fraction of HOz. or of some similar radicals formed during the course of the enzyme action. The present studies with chick liver xanthine dehydrogenase, which reacts only very slowly with oxygen, were undertaken to test this hypothesis. It was also found that there is a nonstoichiometric rehtionship between the DBA++l reduced in the course of the dehydrogenase action and the uric acid formed. The quantity of DBA++ reduced is related to the uric acid produced in such a way that it might be assumed that the major production of uric acid is accomplished through a chain mechanism. Careful examination of possible kinetic schemes shows, however, that so long as the oxygen tension is high, it is necessary to assume a chain or radical mechanism only for the “irreversible” reduction of DBA+f, the uric acid presumably being produced by a twoelectron transfer to DBAff, giving rise to a compound that is very rapidly reoxidiaed. This compound is probably lO,lO’dimethyl-9-acridanyl-9’-acridylium ion, but it has not been positively identified. In the case of milk xanthine oxidase, the reoxidation by oxygen of the reduced enzyme is so rapid that there is little reduced flavoprotein to sustain a chain reaction that we have