Glutathione, Cysteine, and D-Penicillamine Role in Exchange of Silver Metal from the Albumin Metal Complex

Abstract

The purpose of this study is to investigate the exchange reaction taking place among the bovine serum albumin (BSA), 5,5'-dithiobis-(2-nitrobenzoic acid (ESSE), reduced glutathione, N-acetylcysteine, D-penicillamine (thiolates), and silver metal (Ag^I). For this purpose, stock solutions of BSA and Ellman's reagent were prepared by dissolving 264 mg of BSA in 5 ml of reaction buffer (0.1 M KH₂PO₄ at pH 7.8) and 23.8 mg of ESSE in 1.0 ml of reaction buffer which were mixed together. Mixture of BSA-Ag^I was prepared in a separate procedure by dissolving 0.17 mg of silver nitrate in 1 ml of reaction buffer and then dissolving BSA (200 mg) in the same solution of silver nitrate. Blocking of Cys-34 of BSA with Ag^I was confirmed by treating different dilutions of BSA-Ag^I (500 μM) solutions with the solutions of ESSE (85 μM) and ES^- (85 μM) and recording the spectra (300-450) with a UV-visible spectrophotometer. The chromatographed Ag^I-modified BSA ((BSA-S)Ag^I)) samples (typically 500 μM) were subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). Ag^I and modified BSA (typically 500 μM each) were treated with these low molecular weight thiolates and allowed to react overnight followed by chromatographic separation (Sephadex G25). The redox reactions of Ag^I-modified BSA with various low molecular weight thiols revealed a mechanically important phenomenon. In the case of reduced glutathione and N-acetylcysteine, we observed the rapid release of a commensurate amount of Ellman's anion, indicating that an exchange has taken place and low molecular weight thiols (RSH) substituted Ag^I species at the Cys-34 of BSA eventually forming disulfide (BSA-SSR) at Cys-34. It can be anticipated from the phase of study involving bovine serum albumin that low molecular weight thiolates (reduced glutathione and N-acetylcysteine) take off Ag^I which are attached to proteins elsewhere in the physiological system, making these toxic metals free for toxic action.