Characterization of Lead-Phytochelatin Complexes by Nano-Electrospray Ionization Mass Spectrometry
News Mar 02, 2012
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-PC(2) allowed the identification of four different complexes observed at m/z 746.10, 952.06, 1285.24, and 1491.20, corresponding to [Pb-PC(2)](+), [Pb(2)-PC(2)](+), [Pb-(PC(2))(2)](+), and [Pb(2)-(PC(2))(2)](+). Their m/z indicated coordination of Pb(II) by PC(2) through the thiol groups of PC cysteine and possibly carboxylic groups. For each of the standard PC(3) and PC(4), two different complexes were observed, corresponding to Pb-PC(3), Pb(2)-PC(3), Pb-PC(4), and Pb(2)-PC(4). 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-PC(2) complexes showed the appearance of the [Zn-PC(2)](+) complexes at m/z 602.05 and the decrease of the [Pb-PC(2)](+) 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.
The article is published online in Frontiers in Microbiology and is free to access.