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

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.