OPTICAL SPECTROSCOPIC AND REVERSE-PHASE HPLC ANALYSES OF HG(II) BINDING TO PHYTOCHELATINS

Optical spectroscopy and reverse-phase HPLC were used to investigate t he binding of Hg(II) to plant metal-binding peptides (phytochelatins) with the structure (gamma Glu-Cys)(2)Gly, (gamma Glu-Cys)(3)Gly and (g amma Glu-Cys)(4)Gly. Glutathione-mediated transfer of Hg(II) into phyt ochelatins 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 (gamma Glu-Cy s)(2)Gly suggested the formation of a single Hg(II)-binding species of this peptide with a stoichiometry of one metal ion per peptide molecu le. The separation of apo-(gamma Glu-Cys)(2)Gly from its Hg(II) deriva tive on a C-18 reverse-phase column also indicated the same metal-bind ing stoichiometry. The UV/visible spectra of both (gamma Glu-Cys)(3)Gl y and (gamma Glu-Cys)(4)Gly 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 ar ound 1.25 and 2.0 Hg(II) ions per peptide molecule, were observed for (gamma Glu-Cys)(3)Gly. These species exhibited specific spectral featu res in the charge-transfer region and were separable by HPLC. Similarl y, two main Hg(II)-binding species of (gamma Glu-Cys)(4)Gly were obser ved 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)-(ga mma Glu-Cys)(4)Gly complexes was resolved under the conditions used fo r HPLC. The overall Hg(II)-binding stoichiometries of phytochelatins w ere similar at pH 2.0 and at pH 7.4, indicating that pH did not influe nce the final Hg(II)-binding capacity of these peptides. The reverse-p hase HPLC assays indicated a rapid transfer of Hg(II) from glutathione to phytochelatins. These assays also demonstrated a facile transfer o f the metal ion from shorter- to longer-chain phytochelatins. The stre ngth of Hg(II) binding to glutathione and phytochelatins followed the order: gamma Glu-Cys-Gly < (gamma Glu-Cys)(2)Gly < (gamma Glu-Cys)(3)G ly < (gamma Glu-Cys)(4)Gly.