The reactivity of various natural and synthetic phenolic estrogens with copper- and iron-containing oxidases was studied (183, 184, 198, 202). Low concentrations of many phenolic estrogens were found to mediate the aerobic oxidation of reduced pyridine nucleotides, ascorbic acid, ferro-cyanide, and ferrocytochrome c catalyzed by phenolases purified from potatoes or mushrooms. These oxidations were inhibited by cyanide but not by catalase; the cyanide inhibition was reversed by cupric ions. Mono-phenolic estrogens exhibited a lag period for the oxidation of DPNH, when these reactions were studied at low enzyme concentrations, whereas the corresponding o-diphenols did not. In the case of estradiol-17β, evidence was obtained that this steroid was hydroxylated ortho to the hydroxyl group in ring A and that the oxidation of DPNH then proceeded according to the following equations:

T. Akazawa and E. E. Conn (J. Biol. Chem., 232:403, 1958) showed that many phenols mediate the oxidation of DPNH by peroxidase in the presence of Mn++ and oxygen. These phenol-dependent oxidations do not require the addition of hydrogen peroxide; they are inhibited by cyanide and by catalase. It was found (198) that low levels of many phenolic estrogens transported hydrogen in this system. But there was no correlation between estrogenic and hydrogen-transporting activity, and some aromatic amines (199) were also found to transport hydrogen under these conditions. The phenols and aromatic amines probably mediate these oxidations by undergoing one electron oxidation to the corresponding free radicals which are, in turn, reduced non-enzymatically by the hydrogen donor. If the estradiol-mediated oxidation of DPNH was carried out in the presence of an excess of the hydrogen donor, then estradiol-17β could be recovered unchanged from the reaction mixture. But upon exhaustion of hydrogen donor, the oxidation product of this phenolic estrogen underwent further changes, including irreversible binding to protein and also dimerization. The peroxidase-catalyzed oxidation of diethylstilbestrol to the corresponding quinone, and the rearrangement of this quinone to isodienestrol, was studied in detail.

V. P. Hollander and his collaborators (J. Biol. Chem., 235:1504, 1960) reported that the large cytoplasmic particles of rat uterus contain an enzyme system that oxidizes reduced pyridine nucleotides in the presence of oxygen, Mn++, and some phenols, including certain phenolic estrogens. The activity of this enzyme system was negligible in the uteri of spayed or immature animals, but it appeared rapidly, soon after the administration of either natural or synthetic estrogens. These findings were confirmed and extended (183, 202). Optimal conditions for the phenol-dependent oxidation of DPNH were established. Evidence was obtained that the uterine enzyme catalyzing these oxidations was a heme-protein which did not seem to be identical with any known cytochrome. The activity of the phenol-dependent oxidase system was increased about 150-fold after dosage of spayed animals with diethylstilbestrol, whereas the DPNH-cyto-chrome c reductase levels and the aerobic oxidation of DPNH in the presence of cytochrome c were increased less than twofold by estrogen administration. The phenol-dependent oxidations are probably catalyzed by the uterine peroxidase first described by E. Stotz and colleagues (J. Biol. Chem., 214:775, 1955). Some degree of purification of this enzyme was achieved by fractionation with ammonium sulfate and by chromatography on ion-exchange resins.