Characterization of Polysulfides, Polysulfanes, and Other Unique Species in the Reaction between GSNO and H2S

Abstract

Glutathione-based products, GS_nX, of the reaction of hydrogen sulfide, H₂S, S-nitroso glutathione, and GSNO, at varied stoichiometries have been analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) and chemical trapping experiments. A wide variety of glutathione-based species with catenated sulfur chains have been identified including sulfanes (GSS_nG), sulfides (GSS_nH), and sulfenic acids (GS_nOH); sulfinic (GS_nO₂H) and sulfonic (GS_nO₃H) acids are also seen in reactions exposed to air. The presence of each species of GS_nX within the original reaction mixtures was confirmed using Single Ion Chromatograms (SICs), to demonstrate the separation on the LC column, and given approximate quantification by the peak area of the SIC. Further, confirmation for different GS_nX families was obtained by trapping with species-specific reagents. Several unique GS_nX families have been characterized, including bridging mixed di- and tetra-valent polysulfanes and internal trithionitrates (GSNHS_nH) with polysulfane branches. Competitive trapping experiments suggest that the polysulfane chains are formed via the intermediacy of sulfenic acid species, GSS_nOH. In the presence of radical trap vinylcyclopropane (VCP) the relative distributions of polysulfane speciation are relatively unaffected, suggesting that radical coupling is not a dominant pathway. Therefore, we suggest polysulfane catenation occurs via reaction of sulfides with sulfenic acids.

Keywords: S-nitroso glutathione; dimedone; high-resolution mass spectrometry; hydrogen sulfide; iodoacetamide; polysulfanes; sulfenic acids; sulfides; vinylcyclopropane.

Scheme 1

Species generated in the reaction between GSNO and Na₂S.

Scheme 2

Structures of electrophilic, nucleophilic and radical traps used in this study.

Figure 1

Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of oxidized glutathione polysulfane species formed in a 1:5 reaction of GSNO with Na₂S at pH 7, with 0.1% formic acid-acetonitrile as a carrier stream. (a) The total ion LC spectra. (b) Normalized selective ion chromatograms (SICs) showing a series of GSS_nSG polysulfanes, n = 0–8, and the separation times of each within the overall LC spectra. (c) Region of the total ion mass spectra showing peaks assigned as shown, to dications. (d) Logarithmic representations of speciation concentrations calculated from the peak area of the respective species in the LC-HRMS spectral analysis of oxidized glutathione polysulfanes in the reaction of one equivalent of GSNO with increasing equivalents of Na₂S under aerobic condition: (i) 0.25, (ii) 0.5, (iii) 1, (iv) 2.5 and (v) 5 equivalents of Na₂S.

Figure 2

(a) Individual SICs glutathione polysulfane acetamide adducts obtained, RSS_nCH₂CONH₂ (n = 0–9) from LC/MS analysis of GSNO (1 mM) with an Na₂S (1 mM) reaction in the presence of IA (5 mM) in iP buffer pH 7 and (b) their corresponding SIC MS.

Figure 3

Logarithmic relative distribution of GSS_nA adducts in (a) aerobic and (b) anaerobic reactions between GSNO and Na₂S (1:1) with IA present at the beginning (filled circle) or added after 1 hr initiation (filled square) as calculated from the peak area of their respective SICs.

Figure 4

Normalized SIC (a) mass spectra of oxidized glutathione polythiosulfonates, GSS_nSO₃H, generated in the aerobic reaction of GSNO (1 mM) with Na₂S (1 mM) in IP buffer at pH 7 and (b) relative distribution as calculated from the peak area of the SICs.

Figure 5

Relative yields of GSS_nD adducts in (a) aerobic and (b) anaerobic reactions between GSNO and Na₂S (1:5 mM) with DH present in situ (squares in a & b) or added after 1 h initiation (circles in a & b) as calculated from the peak area of the SICs.

Scheme 3

Sulfenic acid tautomers.

Figure 6

Mass spectra of sulfanedithiols derived from sulfinyls of (H₂SO) and GS(O)H in the reaction of GSNO (1 mM) and Na₂S (1 mM) in the presence of IA (5 mM) in iP buffer, pH 7.

Scheme 4

Structural isomers of trithionitrates.

Figure 7

(a) Normalized SICs of glutathione sulfonamide GSS_nNH₂ in the reaction of GSNO (1 mM) with Na₂S in iP buffer at pH 7 and (b) relative distribution as calculated from the peak area of the SICs for reactions at Na₂S ratios of (square) 0.25, (circle) 0.5, (rectangle), 1 mM.

Figure 8

MS of GSS_nNA₂ in the reaction GSNO (1 mM) and Na₂S (1 mM) in the presence of IA (5 mM) in iP buffer, pH 7.

Figure 9

Relative distributions (A) and natural logarithm of relative distributions (B) of DH trapped GSS_nD, acetamide trapped GSS_nA, and oxidized GSS_nG species determined from reactions of GSNO (1 mM) with Na₂S (1 mM): (Squares) in the presence of DH (50 mM) and IA (50 mM); (triangles) in the presence of IA only; (circles) in the absence of DH and IA in iP buffer at pH 7.

Scheme 5

Proposed sequence of speciation

Figure 10

Relative distributions of DH trapped GSS_nD, acetamide trapped GSS_nA, and oxidized GSS_nG species determined from LCMS study of reaction of GSNO (1 mM) with Na₂S (1 mM) and radical clock vinylcyclopropane (5 mM) in iP buffer at pH 7; (filled squares) in the presence of DH (5 mM), (filled circles) in the presence of IA (5 mM); (open squares) in the presence of DH and vinylcyclopropane, (open circles) in the presence of IA and vinylcyclopropane, and (filled diamond) in the presence of only vinylcyclopropane.

Figure 11

Relative distributions of GSS_n-pent-2-ene (GSS_nP), GSS_nO-pent-2-ene (GSS_nOP) determined from LCMS study of reaction of GSNO (1 mM) with Na₂S (1 mM) and radical clock vinylcyclopropane (25 mM) in iP buffer at pH 7; (squares) in the presence of vinylcyclopropane, (up-pointing triangles) in the presence of DH and vinylcyclopropane, (circles) in the presence of IA and vinylcyclopropane, and (down-pointing triangles) in the presence of IA, DH, and vinylcyclopropane.