CATALYSIS OF THE DEALKYLATION DEBENZYLATION OF PHENOXAZONE ETHERS BY HEMOGLOBIN IN THE ABSENCE OF PEROXIDES - IMPLICATIONS FOR INVESTIGATIONS OF EMBRYONIC BIOTRANSFORMATION/

Investigations of catalysis of the O-dealkylation and O-debenzylation of phenoxazone (resorufin) ethers in human and rodent embryonic tissue homogenates indicated that, with few exceptions, each conceptal tissu e investigated contained enzymes capable of catalyzing each of the rea ctions under study. All observable reactions exhibited NADPH dependenc e and strong inhibition by carbon monoxide, ketoconazole, alternate el ectron accepters, and by hypoxic incubation conditions; but, they were not strongly inhibited by several other classical cytochrome P450 (P4 50) inhibitors. Cyanide, azide, superoxide dismutase/catalase, and glu tathione/glutathione peroxidase each also failed to inhibit the reacti ons significantly, Subcellular fractionation experiments revealed that cytosolic fractions contained a preponderance of the observable monoo xygenase activities. Attempts to identify components responsible for t he cytosolic catalytic activity indicated that cytosolic nitric oxide synthases did not contribute significantly, Column fractionation of th e cytosol indicated that significant catalytic activity coeluted with fractions containing hemoglobin (Hgb), and experiments with purified H gb as enzyme source showed that Hgb would catalyze all reactions under study at very slow rates in the absence of added reductases or peroxi des. Additions of either reductases or peroxides, however, resulted in marked increases in rates of Hgb-catalyzed reactions, Further investi gations strongly suggested that virtually all dealkylation or debenzyl ation of phenoxazone ethers catalyzed by embryonic cytosolic fractions could be accounted for by the presence of Hgb in those fractions. Con ceptal microsomal fractions, however, exhibited definitive, P450-depen dent monooxygenase activities attributable to specific individual, ide ntifiable P450 isoforms.