Optical spectroscopic and reverse-phase HPLC analyses of Hg(II) binding to phytochelatins

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Abstract
Optical spectroscopy and reverse-phase HPLC were used to investigate the binding of Hg(II) to plant metal-binding peptides (phytochelatins) with the structure (γGlu-Cys)2Gly, (γGlu-Cys)3Gly and (γGlu-Cys)4Gly. Glutathione-mediated transfer of Hg(II) into phytochelatins and the transfer of the metal ion from one phytochelatin to another was also studied using reverse-phase HPLC. The saturation of Hg(II)-induced bands in the UV/visible and CD spectra of (γGlu-Cys)2Gly suggested the formation of a single Hg(II)-binding species of this peptide with a stoichiometry of one metal ion per peptide molecule. The separation of apo-(γGlu-Cys)2Gly from its Hg(II) derivative on a C18 reverse-phase column also indicated the same metal-binding stoichiometry. The UV/visible spectra of both (γGlu-Cys)3Gly and (γGlu-Cys)4Gly at pH 7.4 showed distinct shoulders in the ligand-to-metal charge-transfer region at 280–290 nm. Two distinct Hg(II)-binding species, occurring at metal-binding stoichiometries of around 1.25 and 2.0 Hg(II) ions per peptide molecule, were observed for (γGlu-Cys)3Gly. These species exhibited specific spectral features in the charge-transfer region and were separable by HPLC. Similarly, two main Hg(II)-binding species of (γGlu-Cys)4Gly were observed by UV/visible and CD spectroscopy at metal-binding stoichiometries of around 1.25 and 2.5 respectively. Only a single peak of Hg(II)–(γGlu-Cys)4Gly complexes was resolved under the conditions used for HPLC. The overall Hg(II)-binding stoichiometries of phytochelatins were similar at pH 2.0 and at pH 7.4, indicating that pH did not influence the final Hg(II)-binding capacity of these peptides. The reverse-phase HPLC assays indicated a rapid transfer of Hg(II) from glutathione to phytochelatins. These assays also demonstrated a facile transfer of the metal ion from shorter- to longer-chain phytochelatins. The strength of Hg(II) binding to glutathione and phytochelatins followed the order: γGlu-Cys-Gly < (γGlu-Cys)2Gly < (γGlu-Cys)3Gly < (γGlu-Cys)4Gly.