Quantitative evaluation of the binding of phenylarsenic species to glutathione, isotocin, and thioredoxin by means of electrospray ionization time‐of‐flight mass spectrometry

Abstract

An attempt was made to quantitatively describe the binding of phenylarsenic species to thiol-containing biomolecules using electrospray ionization mass spectrometry (ESI-MS). The extent of the reactions of phenylarsine oxide (PAO) with the peptides glutathione and isotocin (ITC) and with the protein thioredoxin resulting in covalent As--S bonds were quantified by deriving the dependence of the corresponding ion signal intensities on the concentration of the reaction products. Problems complicating a quantitative evaluation of the mass spectra, such as signal suppression effects, were critically evaluated. Equilibrium constants for condensation reactions as well as formation constants for noncovalent associations were calculated by means of ESI-MS signal intensities. The comparison of the reaction of PAO with different thiol reactants revealed the highest binding affinity for ITC followed by thioredoxin and a lower affinity to glutathione. Possibly, the intramolecular formation of RS-As(C(6)H(5))-SR occurring in case of ITC and thioredoxin is favored over the intermolecular product involving two molecules glutathione even though the molecular mass of glutathione (307 g mol(-1)) is much smaller than that of ITC (966 g mol(-1)) and thioredoxin (11 688 g mol(-1)). A similar binding affinity for trivalent (K approximately 1.6 x 10(-3) l micromol(-1)) and pentavalent (K approximately 1.6 x 10(-3) and 1.0 x 10(-3) l micromol(-1)) arsenic species was found for the formation of a noncovalent complex of glutathione with different phenylarsenic compounds.

Optimization of peptide and protein separation with a monolithic reversed-phase column and application to arsenic-binding studies

“…10 mg mL −1 stock solutions of peptides and proteins in deionized water were stored at −18 • C. For HPLC measurements, 0.5 mg mL −1 and 0.1 mg mL −1 dilutions for single component measurements and for mixed samples, respectively, were prepared freshly in deionized water. According to our previous work [6,16], for arsenic binding studies, the biomolecules were reduced with tris(carboxyethyl)phosphine (TCEP, purchased as 0.5 M hydrochloride solution from Sigma-Aldrich). For mixed samples, the peptides and proteins were incubated for 10 min with a 10-fold molar excess of the reducing agent related to the sum concentration of all biomolecules present in the sample in order to ensure a complete reduction and therewith to obviate an impact of the reduction degree on the arsenic-thiol reaction.…”

“…In former studies [5,6,16], condensation reactions of the organic arsenic compound phenylarsine oxide with different cysteinecontaining peptides and proteins in their reduced state were observed by ESI-MS. Since the formed arsenic-sulfur bindings are very stable in contrast to bindings of other arsenic species, reactions with PAO can serve as an easily manageable probe for method development concerning the analysis of arsenic-binding biomolecules.…”

“…However, mass spectrometric binding studies are complicated by some drawbacks such as the transfer of the analytes in the gas phase, competing effects in the ionization process, and unknown ionization efficiencies for protein-ligand complexes. In comparative investigations, large differences in binding constants determined by means of the SEC-ESI-MS coupling and by direct ESI-MS measurements without chromatographic coupling were observed [5,6]. Because reversed phase liquid chromatography exhibits a much higher chromatographic resolution for peptide and protein separations, this type of separation techniques was now implemented for qualitative and quantitative analysis of arsenicbinding biomolecules.…”

“…Because reversed phase liquid chromatography exhibits a much higher chromatographic resolution for peptide and protein separations, this type of separation techniques was now implemented for qualitative and quantitative analysis of arsenicbinding biomolecules. Cysteine-containing peptides and proteins were chosen for which condensation reactions took place with an organic arsenic compound under reducing conditions [5,6]. The nonapeptides vasotocin (Vtc, M = 1.05 kDa, pI ≈ 9.84), vasopressin (Vpr, M = 1.084 kDa, pI ≈ 9.84), and oxytocin (Otc, M = 1.007 kDa, pI ≈ 6.01) possess one structure-stabilizing disulfide bridge each, whereas the small protein aprotinin (Apr, M = 6.5 kDa, pI = 10.5) exhibits three constitutive disulfide bridges.…”

Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

“…Trivalent arsenic compounds react with thiol groups of biomolecules and inhibit their biological activity . Reactions of the warfare degradation product PAO containing trivalent arsenic with several cysteine‐containing peptides and proteins were elucidated in our previous works using electrospray ionization mass spectrometry (ESI‐MS) . Bindings of arsenic compounds to biomolecules can be easily detected using ESI‐MS due to the change of molecular mass .…”