Probing the coordination behavior of Hg2+, CH3Hg +, and Cd2+ towards mixtures of two biological thiols by HPLC-ICP-AES

摘要

Hepatocyte cytosol contains a multitude of proteins, but also comparatively high concentrations of l-glutathione (GSH, ～ 5.0 mM) and l-cysteine (Cys, ～ 0.5 mM). Since Hg2+, CH3Hg+ and Cd 2+ have a high affinity for thiols, their coordination to these thiols is likely involved in their intracellular transport. The comparative coordination behavior of these metal species towards mixtures of Cys and GSH, however, has not been studied under near physiological conditions. To probe these toxicologically relevant interactions, each metal species was separately injected onto a C18-HPLC column (37 °C) that had been equilibrated with phosphate buffered saline (PBS) that contained 5.0 mM GSH (mobile phase) and detected with an inductively coupled plasma atomic emission spectrometer. The incremental increase of the Cys concentration in the mobile phase (in 0.5 or 1.0 mM steps) up to 10 mM followed by the chromatography of each metal species decreased the retention of Hg2+ and CH 3Hg+ albeit in a different manner. This behavior was rationalized in terms of the replacement of hydrophobic GS-moieties coordinated to each mercurial by less hydrophobic Cys-moieties. In contrast, a Cd-peak eluted close to the void volume with all investigated mobile phases. Using X-ray absorption spectroscopy, the Cd-compound that eluted with a PBS-buffer that contained 5.0 mM GSH was structurally characterized as tetrahedral (GS) 4Cd. Thus, the in vivo formation of (GS)4Cd must be considered and HPLC-ICP-AES is identified as a useful tool to probe dynamic bioinorganic processes which involve the interaction of a metal ion with multiple ligands under physiologically relevant conditions.