(b) Mueller (88) found that the incorporation of formate into the protein of isolated uterine horns was stimulated by the in vitro addition of either 2-hydroxy- or 4-hydroxy-estradiol-17β, but not by estradiol-17β itself.

(c) o-Hydroxylated derivatives are among the metabolites of ovarian estrogens. Thus 2-methoxyestrone (70) and 2-methoxyestriol (31) have been isolated from the urine of women.

We observed (138, 139) that phenolases isolated from white potatoes and edible mushrooms catalyzed the oxidation of a number of hydrogen donors in the presence of very low concentrations of many phenolic estrogens. The potato enzyme was studied most thoroughly, and the main findings may be summarized as follows. In manometric experiments conducted at pH 7.4, the oxidation of ascorbic acid, DPNH, and TPNH by crude aqueous extracts of potato peel was accelerated as much as one-hundredfold by 10-5M estradiol-17β or hexestrol. The action of these phenolic estrogens was apparent when DPNH or TPNH were added as such, or generated from low levels of the oxidized nucleotides by the respective action of yeast alcohol or glucose-6-phosphate dehydrogenases. The oxidations proceeded to completion with the consumption of one atom of oxygen per mole of DPNH oxidized. The oxidation of reduced pyridine nucleotides was also measured spectrophotometrically at 340 mu. Using the latter assay method, it was found that the estradiol-stimulated oxidation of DPNH paralleled the phenolase activity (oxidation of a mixture of tyrosine and 3,4-dihydroxy-phenylalanine) during purification of the enzyme. With more purified enzyme preparations, the oxidation of DPNH or TPNH was negligible in the absence of catalytic concentrations of an appropriate phenol. The oxidation of ferrocytochrome c was also accelerated by trace levels of estradiol-17β. The optimum pH for these estradiol-mediated oxidations was in the vicinity of pH 7. The reactions were inhibited completely by 10-3 sodium cyanide, and the cyanide inhibition could be reversed by the addition of Cu++ ions. Neither CuSO4, nor crystalline hemocyanin, nor a soluble phenolase prepared from spinach leaves had any catalytic action for these estradiol-mediated reactions. The phenolase-catalyzed oxidation of DPNH mediated by estradiol-178 was uninfluenced by the addition of crystalline catalase.

Only those estrogens bearing at least one free phenolic hydroxyl group were found to be capable of transporting hydrogen under these conditions. Thus, the carrier activity of estradiol-17a and -17β, 17-deoxyestradiol, estrone, and estriol is virtually the same, whereas 3-deoxyestradiol-17a or -17β are completely inert. The action of bisdehydrodoisynolic acid disappears when the compound is O-methylated. Many hydroxylated stilbenes, equilin and equilenin, and also genistein were active carriers. None of the nonaromatic steroid hormones tested in this system were found to transport hydrogen, e.g., progesterone, testosterone, cortisone, 1- and 4-estrene-3,17-dione, l,4-androstadiene-3,17-dione, and 5-androstene-36,178-diol. Similarly, a number of ring A substituted steroidal estrogens, such as 2- and 4-nitroestrone, 68-hydroxyestradiol-176, and l-methylestradiol-176, were inactive. The concentrations of various phenolic estrogens required to induce 50% of the maximal rate of oxidation of DPNH at pH 7.4 are depicted in Table VIII.

Table VIII. Affinity Of Potato Phenolase For Phenolic Estrogens And Other Phenols*

* Reactions carried out in a final volume of 3.0 ml. at 25° C. Each vessel contained 250 umoles sodium phosphate buffer pH 7.4; 0.3 (imole DPNH; 100 ug. potato phenolase and suitable amounts of phenols in 0.01 ml. dioxane. The results were corrected for the very small rate of oxidation of DPNH in the presence of dioxane, but in the absence of phenol. Wavelength: 340 mµ.

b Molar concentration of phenol required to induce 50% of the maximal rate of oxidation of DPNH. The latter was determined from the linear reaction rate which was established at the end of the lag period.

In spectrophotometric experiments performed with low enzyme concentrations, it was observed that a marked induction period occurred when estradiol-17β, and other monophenolic estrogens, were used as carriers. The extent of this lag, as well as the final maximal rate of oxidation of DPNH was, within limits, proportional to the quantity of enzyme present. Figure 7 shows that no such induction period was demonstrable with 4-hydroxyestradiol-178, one of the o-diphenolic forms of this estrogen. This compound was kindly provided by Dr. G. C. Mueller. These findings were in accord with an initial hydroxylation of the estradiol-17β to an o-diphenolic derivative (presumably reflected by the induction period), which could then be oxidized to the corresponding o-quinone. The quinone would then be reduced by the hydrogen donor. This mechanism was further supported by the demonstration that estradiol-17β in low concentrations (6 X 10-5M) was converted into derivative(s) which chromatographed on paper closely to 2- and 4-hydroxyestradiol-17β in a heptane-methanol solvent (22) under conditions where estradiol-17β mediated the oxidation of DPNH by potato phenolase.

A pronounced lag period was also observed when hexestrol and diethylstilbestrol were used as carriers for the oxidation of reduced pyridine nucleotides, but not with certain corresponding o-hydroxylated derivatives, e.g., 3- hydroxyhexestrol. However, the possible transformation of hexestrol under conditions where this substance transported hydrogen in these phenolase-catalyzed systems was not examined, and the mechanism of the carrier action of hydroxylated stilbenes remains to be clarified. Any bearing of these model reactions with plant phenolases on the metabolism or mode of action of estrogens in animal tissues is purely a matter of conjecture. The distribution of phenolases among various mammalian cells appears to be rather narrow; they are undoubtedly present in skin and neoplasms thereof

Fig. 7. Oxidation of DPNH by purified potato phenolase in the presence of estrogenic phenols.

The reaction systems contained in a final volume of 3.0 ml.: ISO umoles sodium phosphate buffer of pH 7.4, 0.32 umole DPNH and 200 ug. potato phenolase. Upper curve: no added phenol. Lower two curves: 15 µg. estradiol-17β and 15 ug. 4-hydroxyestradiol-17β respectively. All cuvettes contained 0.01ml. dioxane. The changes with time of the optical density at 340 mu are shown. Temperature 25"C.

(76), and perhaps in adrenal medulla (131) and in kidney (4). It is tempting to consider that the interaction with phenolases in skin might account for the dramatic effects of estrogens on cutaneous pigmentation (23). The mechanism of the biosynthesis of 2-methoxyestrone (70) and 2-methoxy-estriol (31) is unknown, but it would seem likely that they are formed by methylation of 2-hydroxy derivatives. It is interesting that the liver microsomal enzyme system which Mueller and co-workers (89, 90, 98) found to hydroxylate estradiol-17β at the 6- and 10-positions did not form either 2-or 4-hydroxylated derivatives of this estrogen. Perhaps phenolases are concerned in the formation of 2-hydrox.ylated derivatives of natural estrogens in animal tissues. Finally, it may be mentioned that there is evidence for the participation of phenolases in the terminal respiratory pathways of some plant tissues (85) and that a variety of phenolic estrogens have been isolated from vegetable sources (9, 15, 26). That some of the plant estrogens may act as hydrogen carriers in plant respiration via phenolase-catalyzed reactions merits further study.