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 mu M) solutions with the solutions of ESSE (85 mu M) and ES- (85 mu M) and recording the spectra (300-450) with a UV-visible spectrophotometer. The chromatographed Ag-I-modified BSA ((BSA-S)Ag-I)) samples (typically 500 mu M) were subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). Ag-I and modified BSA (typically 500 mu M 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.