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

Glutathione-based products, GSnX, 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 (GSSnG), sulfides (GSSnH), and sulfenic acids (GSnOH); sulfinic (GSnO2H) and sulfonic (GSnO3H) acids are also seen in reactions exposed to air. The presence of each species of GSnX 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 GSnX families was obtained by trapping with species-specific reagents. Several unique GSnX families have been characterized, including bridging mixed di- and tetra-valent polysulfanes and internal trithionitrates (GSNHSnH) with polysulfane branches. Competitive trapping experiments suggest that the polysulfane chains are formed via the intermediacy of sulfenic acid species, GSSnOH. 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.


S9.
Reaction of GSNO with H2S with and without NaS2O3 S10. Mass spectra of reduced GSSnH identified in the reaction of GSNO with H2S. S11. LC-MS/MS of GSSnH products S12. LC-MS/MS of GSSnSO3H products S13. MS comparison of GSSH and GSO2H in aerobic reactions S14. LC-MS/MS of GSSnSO2H products S15. LC-MS/MS of GSSnNH2 products S16. LC-MSMS of GSNSnA2 products S1. LCMS of oxidized polysulfides in MeOH with 1 % formic acid mobile phase

S3. Reaction of GSNO with H2S in carbonate buffer at pH 10.
High pH reactions of GSNO with H2S. The reaction solutions were allowed to stand for an hour before LC/MS analysis. For temperature dependent studies the reaction solutions were incubated at 37 O C in Excella E24 incubator.

S5. Reaction of GSNO with H2S gas
Reaction of GSNO or GSSG with H2S(g). As a control reaction. gaseous H2S was generated by addition of concentrated HCl to Na2S under inert N2 purge which was bubbled directly through an aqueous solution of GSNO or GSSG in iP buffer pH 7. The reaction solutions were allowed to stand for an hour before LC/MS analysis. Figure S5. Orbitrap LCMS study of reaction of GSNO (1mM) with H2S (gas) generated from Na2S-HCl mixture in iP buffer at pH 7. (Top) SIC of glutathione polysulfides (oxidized) species and (Bottom) their corresponding mass spectra.

Reactions with elemental Sulfur(s). To reaction solutions of GSH (1 mM) or GSSG (1 mM)
were added elemental sulfur (solid) in iP buffer, pH 7 and allowed to stand for 24 h. Figure 6. SICs of oxidized glutathione polysulfides (GSSxSG) from the Orbitrap LCMS analysis of reaction of (A) 1 mM of GSSG, (B) 1 mM of GSH with excess S in iP buffer at pH 7 and (C) relative distribution of the glutathione polysulfide species.

Reactions of GSNO/H2S in presence and absence of S(s).
To the reaction solutions of GSNO (1 mM) and Na2S (5 mM), were added elemental sulfur (solid) in iP buffer, pH 7 and allowed to stand for 24 h. Figure S7. Relative distribution of the glutathione polysulfide species calculated in the presence (red) and absence (black) of S with GSNO (1 mM) and Na2S (5 mM) in iP buffer at pH 7.
Reactions of sulfite and thiosulfate with GSH. 5 mM of GSH was mixed either with 10-fold excess Na2SO3 or Na2S2O3 in iP buffer pH 7. The reaction solutions were allowed to stand overnight before LC/MS analysis. Figure S8. Orbitrap LCMS study of reaction of GSH (1 mM) with Na2S2O3 (5 mM) in iP buffer at pH 7. Top: SIC of glutathione polysulfides (oxidized) species. Bottom: their corresponding mass spectra.