Transient Free Radical Forms of Hormones: Epr Spectra from Iodothyronines, Indoles, Estrogens, and Insulin.

In a preceding communication' we presented electron paramagnetic resonance (EPR) evidence for the existence of labile free radical forms of catecholamine hormones and adrenochrome following rapid oxido-reductions with inorganic reagents in vitro. Although the relevance of these demonstrations to the primary physiochemical interactions of hormones in vivo was left open, generalization of the findings to other hormones emphasizes the possible involvement of free radical reactions. This report concerns similar EPR studies of free radicals formed from small hormone molecules of the thyronine, indole, and estrogenic steroid classes and from one protein hormone, insulin. Experimental.-As in the previous article,' an improved high velocity continuous flow apparatus adapted for EPR spectroscopy was used. This has been described elsewhere.2' 3 Spectra usually were scanned in about 3 sec, but some scans required 5-6 sec. Volume flow rates ranged from 4 to 17 ml/sec with reactants kept equimolar at concentrations of 1-2 X 10-2 M. In the figures, magnetic field increases from left to right. The uncertainty in gvalue is approximately 4-0.001. Results and Discussion.-Thyronines: Stimulation by thyroxine analogues of horseradish peroxidase action on a number of donor substrates led Klebanoff to a broad implication of free radical intermediates by analogy with previous analyses of other peroxidase reactions.4 A subsequent study of similar stimulation of peroxidase oxidation of sulfite led to an explicit citation of thyroxine free radicals as probable reaction intermediates.5 The presence of free radicals of thyroxine or of its analogues also has been inferred from the effect of thyroxine on copper-stimulated ascorbic acid oxidase,6 from structure-activity correlations of the inhibition of thyroxine degradation by microsonial systems,7 anid from analysis of light-stimulated deiodinationI of thyroxine by liver homogenates.A A protein-bound quinoid free radical of thyroxine was proposed to explain the behavior of various in vitro oxidation reactions of the hormone and the mietabolisnm of several of its analogues.9 Despite these speculations, explicit confirmation of thyroxine free radicals apparently has not been made previously. Other hormonal radicals we investigated turned out to be very short-lived,' perhaps explaining the lack of EPR data heretofore. However, the thyroxine-like iodinated thyronines proved to be exceptions to this general finding: although their free radicals formed "instantly," they were remarkably stable (minutes to hours) at alkaline pH's. The spectrum of thyroxine stoichiometrically oxidized in alkaline solution by one electron equivalent of ferricyanide or other oxidant (Fig. 1) was typical of the halogenated thyronines we examined. Triiodothyronine and triand tetraiodinated acetic and propionic acid derivatives manifested EPR spectra essentially identical