title: Optical spectroscopic and reverse-phase HPLC analyses of Hg(II) binding to phytochelatins.
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 (gammaGlu-Cys)2Gly, (gammaGlu-Cys)3Gly and (gammaGlu-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 (gammaGlu-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-(gammaGlu-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 (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly at pH 7.4 showed distinct shoulders in the ligand-to-metal charge-transfer region at 280-290 mm. 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 (gammaGlu-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 (gammaGlu-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)-(gammaGlu-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: gammaGlu-Cys-Gly<(gammaGlu-Cys)2Gly<(gammaGlu-Cy s)3Gly<(gamma Glu-Cys)4Gly.
authors.affiliation: Environmental Toxicology Graduate Program, Entomology Department, University of California, Riverside, 92521, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Mehra
authors.forename: J
authors.initials: J
authors.lastname: Miclat
authors.forename: V R
authors.initials: VR
authors.lastname: Kodati
authors.forename: R
authors.initials: R
authors.lastname: Abdullah
authors.forename: T C
authors.initials: TC
authors.lastname: Hunter
authors.forename: P
authors.initials: P
authors.lastname: Mulchandani
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005978:Glutathione / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism; D017669:Mercury Compounds / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D008668:Metallothionein / Q000378:metabolism; D008670:Metals / Q000378:metabolism / Q000633:toxicity; D054811:Phytochelatins; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D010944:Plants / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013053:Spectrophotometry
chemical_list: D017669:Mercury Compounds; D008667:Metalloproteins; D008670:Metals; D010940:Plant Proteins; D008668:Metallothionein; D054811:Phytochelatins; D005978:Glutathione