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

Abstract

Phenylarsenic-substituted cysteine-containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure-stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI-MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic-bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α-lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ß-lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic-sulfur bindings were determined from direct injection ESI-MS measurements than from LC-ESI-MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI-MS and LC-ESI-MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α-lactalbumin > thioredoxin. Kinetic investigations by LC-ESI-MS yielded a partial reaction order of 2 for vasopressin, Lys and α-lactalbumin and corresponding half-lives of 0.93, 2.56 and 123.5 min, respectively.

Arsenic Binding to Proteins

“…For example, a stability constant ( K a = 1/ K d ) of 2 × 10 6 was determined with ITC for the 1:3 arsenite complex with GSH, for which unusually high values of 2 × 10 33 and 1 × 10 32 were reported by Rey et al on the basis of their near-UV absorption and potentiometric data, respectively. The binding affinities between arsenic and several thiol-containing biomolecules were also determined with ESI-MS by Schmidt and co-workers, ,− but their values were inconsistent. One issue with quantifying both the unbound and bound species using ESI-MS is that these species may have different ESI-MS responses and their intensities may not be calibrated against a single species.…”

Sulfur K-edge X-ray absorption spectroscopy and time-dependent density functional theory of arsenic dithiocarbamates

“…1 As exposure from contaminated food and water remains a critical problem in many parts of the world, and its high toxicity and known carcinogenic effects are attributed in part to its affinity for sulfur-containing biochemical groups found in the body. [2][3][4][5] Most notably, trivalent As binds to the thiol group in cysteine, 2,[6][7][8][9][10][11] which is detrimental because of its presence in protein active sites and DNA repair enzymes. Consequently, As can disrupt biochemical function at these locations by inhibiting enzymatic activity and provoking structural rearrangements that contribute to protein degradation.…”