AUTHOR=Scheidegger Christian , Suter Marc J., Behra Renata , Sigg Laura TITLE=Characterization of Lead–Phytochelatin Complexes by Nano-Electrospray Ionization Mass Spectrometry JOURNAL=Frontiers in Microbiology VOLUME=3 YEAR=2012 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2012.00041 DOI=10.3389/fmicb.2012.00041 ISSN=1664-302X ABSTRACT=

The role of phytochelatins (PC_n, metal-binding oligopeptides with the general structure (γGlu-Cys)_n-Gly (n = 2–11) in metal detoxification is assumed to be based on immobilization of metals, which prevents binding of metals to important biomolecules. Although induction of phytochelatin synthesis has often been observed in algae upon exposure to metals, direct evidence for binding of the inducing metal to phytochelatins is scarce. In this study, a nano-electrospray ionization mass spectrometry (nano-ESI-MS) method is developed for identification and characterization of Pb(II)–PC_n and Zn(II)–PC_n complexes. Complexes of Pb(II) with standard PC_n (n = 2–4; 0.25 mM Pb(II) and 0.5 mM PC_n) were examined by nano-ESI-MS with respect to their stoichiometry. Pb–PC_n mass spectra indicated the presence of the [M + H]⁺ peak of PC_n and complexes with various stoichiometries. Analysis of Pb–PC₂ allowed the identification of four different complexes observed at m/z 746.10, 952.06, 1285.24, and 1491.20, corresponding to [Pb–PC₂]⁺, [Pb₂–PC₂]⁺, [Pb–(PC₂)₂]⁺, and [Pb₂–(PC₂)₂]⁺. Their m/z indicated coordination of Pb(II) by PC₂ through the thiol groups of PC cysteine and possibly carboxylic groups. For each of the standard PC₃ and PC₄, two different complexes were observed, corresponding to Pb–PC₃, Pb₂–PC₃, Pb–PC₄, and Pb₂–PC₄. The measured isotopic patterns were for all complexes identical to the theoretical isotopic patterns. Addition of Zn(II) (0.125–5 mM) to previously formed Pb–PC₂ complexes showed the appearance of the [Zn–PC₂]⁺ complexes at m/z 602.05 and the decrease of the [Pb–PC₂]⁺ peak. These findings corroborate the postulated Pb–PC complexes from a previous study using size exclusion chromatography of PC extracted from algae, as well as the concurrent formation of Pb–, Zn–, and Cu–PC complexes in algae.