Rhodanese-catalyzed reduction of thiosulfate by reduced lipoic acid.

by a double displacement mechanism (1, 2). Thiosulfate and organic thiosulfonates will act as sulfur donors for the enzyme (3). Cyanide ion, SOS=, and the organic sulfinates will accept sulfur from the enzyme-sulfur compound (1, 4, 5). In this study, crystalline beef liver rhodanese has been found to catalyze the reduction of SSOp to HSand SOf. Several powerful reducing agents, such as NaBH4 and Na&O+ could be utilized as substrates for the enzymic reaction. In the range of physiologically accessible oxidation-reduction potentials, however, only reduced lipoate and lipoamide were reactive. Rhodanese did not utilize DPNH, TPNH, cysteine, GSH, or formaldehyde for SSOd reduction although these reactions would be exergonic. Evidence was obtained indicating that the enzymic reaction with lipoate or lipoamide is stereospecific. Crystalline beef liver rhodanese, prepared by the method previously described (6), was used in all enzymic studies. DLLipoic acid was purchased from the California Corporation for Biochemical Research. Reduced nn-lipoate was prepared by NaBH4 reduction in a buffer containing sodium phosphate and sodium tetraborate, both at 0.05 M, pH 8.5. m-Lipoamide was purchased from the Aldrich Chemical Company. The use of a Heyrovsky recording polarograph with a dropping mercury electrode permitted measurement of the concentrations of reduced lipoate, HS-, and SSOa=, which display well defined anodic diffusion currents, and oxidized lipoate, which has a cathodic diffusion current at the same half-wave potential as the anodic wave of reduced lipoate (-0.67 volt versus the saturated calomel electrode) .l The HSE+” (-0.66 volt) is too close to that of the lipoate to be resolved but quantitation of all the components of the wave is possible by algebraic addition. The SSOd El ( -0.12 volt) is far enough from the composite lipoate-sulfide wave to permit good measurement of SS03= concentration. When the enzymic reaction was run in the polarograph cell, the disappearance of reduced lipoate and SSOb and the appearance of oxidized lipoate and HScould be followed throughout the course of the reaction (Fig. 1). In the absence of rhodanese, no reaction occurred. Lipoamide could be used in place of lipoate in the enzyme-catalyzed reaction. The data presented in Fig. 1 establish the stoichiometry of the following reaction: