Mass spectrometric evidence for different complexes of peptides and proteins with arsenic(III), arsenic(V), copper(II), and zinc(II) species

Abstract

Trivalent and pentavalent arsenic were incubated with sulfur-containing amino acid, peptide and protein solutions both as organic compounds (phenylarsine oxide, phenylarsonic acid, dimethylarsinic acid, monomethylarsonic acid) and as inorganic compounds (arsenite, As(III), and arsenate, As(V)). After incubation of phenylarsine oxide solutions with cysteine and glutathione the mass spectra showed a covalent bond between arsenic and sulfur, which was stable at both acidic and neutral pH values. The mass spectra were dominated by monovalent ions at m/z 272 for cysteine samples and at m/z 458 for glutathione samples. Based on these masses the ionic structures could be ascribed to either fragment ions of the covalent arsenic-sulfur complexes or to other arsenic-bonding sites presumably at the amino group. Interestingly, under the same conditions no interactions of inorganic arsenite or arsenate could be measured. In the presence of added Cu(2+) ions all mass signals caused by a reaction of phenylarsine oxide with glutathione disappeared. In these mass spectra only the oxidised form of glutathione (GSSG) was found because of the redox activity of Cu(II). For the model protein lysozyme, no interactions with arsenic could be detected, whereas definite Cu- and Zn-lysozyme complexes with a stoichiometry of 1:1 and 2:1 for Zn(2+) ions and Cu(2+) ions, respectively, were observed. In contrast, for thioredoxin a bonding of As that depended on the concentration of the disulfide-reducing agent tris(2-carboxyethyl) phosphine was demonstrated. For three different phenylarsonic acids and for dimethylarsinic acid that all contain pentavalent arsenic, complexes with glutathione appeared in the mass spectra, which can be attributed to non-covalent interactions or to a covalent bond caused by an additive reaction. The optimisation of the experimental conditions necessary for the mass spectrometric analysis of the interactions of the arsenic species with peptides and proteins is described and the obtained mass spectra that provide information on the kinds of bonds are discussed.

CZE of Sulfur-Containing Amino Acids and Peptides and Its Application to the Quantitative Study of Heavy Metal-Caused Thiol Oxidations

“…The oxidation potential of Zn(II) (E Zn(0)/Zn(II) h = -0.7628 V) is not sufficient to oxidize GSH (E GSH/GSSG h = -0.240 V). This finding agrees with mass spectrometric Cu and Zn binding studies of GSH [9]. The standard redox potential of the Ni(0)/Ni(II) redox pair (E Ni(0)/Ni(II) h = -0.236 V) resembles the potential of GSH/GSSG.…”

“…Further, GSH serves as precursor for the biosynthesis of metal-chelating polypeptides involved in metal detoxification and homeostasis [8]. Instead of a direct binding, metal ions characterized by a large oxidation potential, such as Cu(II) oxidize GSH to GSSG [9]. A large number of studies demonstrated that the GSH/GSSG ratio can be considered as a reliable index of the cellular redox state.…”

“…Since physiological conditions should be approximated to implement the metal-biomolecule interaction studies, CZE separations of biological thiols in an acidic [4,10] or alkaline [17] pH range should be avoided. Moreover, higher proton activities compete with metal cations for reaction sites of biomolecules [9]. In the presented method development, a compromise between an about neutral pH value in the separation medium and a high separation power should be found.…”

Review of arsenic speciation, toxicity and metabolism in microalgae

“…Gluthione, PCs and its derivatives are considered to be the most important classes of metal-chelating polypeptides, which contain thiol groups that bind readily with arsenic species (Hall 2002;Schmöger et al 2000;Schmidt et al 2007). These peptides can be found in many higher plants like Holcus lanatus, Cytisus striatus and Pteris vittata Hartley-Whitaker et al 2002;Schmöger et al 2000;Zhao et al 2003) and fungi (Kneer and Zenk 1992) and may be partitioned inside vacuoles to facilitate appropriate control of the cytoplasmic concentration of heavy metal ions (Cobbett and Goldsbrough 2002).…”

Technical Note: Effects of Arsenate (AS5+) on Growth and Production of Glutathione (GSH) and Phytochelatins (PCS) inChlorella Vulgaris

“…The most important classes of metal-chelating polypeptides are glutathione (GSH) and its derivative forms, phytochelatins (PCs) which contain thiol groups that bind readily with As species (Schmidt et al 2007). These peptides can be found in microalgae, related eukaryotic photosynthetic organisms, and some fungi (Perales-Velaet et al 2006) as organometallic complexes.…”