Monobromobimane

H₂S concentrations in the plasma and hearts were measured as previously described with some modifications (Tan et al., 2017 ). First, snap-frozen cardiac tissue was homogenized and centrifuged (12,000 rpm, 20min, 4°C). The protein content in supernatants was measured by using the BCA reagent. Plasma was thawed and mixed before assay. Thirty microliter of samples (plasma or supernatant) was spiked with 80μL monobromobimane (Sigma–Aldrich) and 10 μL of 0.1% ammonia. After being agitated for 60 min at room temperature, the reaction was stopped by adding 10 μL of 20% formic acid. Then, the mixture was subjected to centrifugation at 15,000rpm for 10min, clear supernatants were collected as samples. H₂S levels were quantified against a standard curve generated by Na₂S (0–40 μmol/L). The plasma H₂S levels were expressed as μmol/L, and the heart H₂S levels were divided by the protein concentrations, which were expressed as μmol/g of protein.

Thiols were extracted from 30mg of plant tissue in the presence of dithiothreitol (DTT; for total glutathione) or N-ethylmaleimide (NEM; for oxidized glutathione) and derivatized with monobromobimane (Sigma) as described previously (Fey et al., 2005 (link)). Glutathione was measured by reverse-phase high-performance liquid chromatography (HPLC) according to Liedschulte et al. (2010) (link). GSH was determined by subtracting GSSG from total glutathione.

H₂S assay was based on a previously published method adapted here for tissue lysates [42 (link)]. First, approximately ~10–20 mg of the tissue samples were disrupted by a dismembrator. Alkylation/lysis was carried out by the addition of 500 µL PBS set to pH 8.0 containing 1 mM monobromobimane (Sigma Aldrich, St. Louis, MO, USA) in a light-protected environment. After a short sonication on ice the solutions were incubated for one hour at 37 °C in the dark. The reaction was stopped by the addition of 50 µL 50% TCA followed by centrifugation at 12,000× g 4 °C for 10 min to remove precipitated proteins. Supernatants were removed and transferred into HPLC vials for measurement, and the remaining pellets were redissolved in 300 µL 4% SDS/0.1 M NaOH for BCA protein assay. Bimane labeled species from the supernatants using 3 µL injection volumes were separated on a Phenomenex Luna C18(2) 250 × 2.0 mm × 3μm column on a Thermo Ultimate 3000 UHPLC system (Thermo Fisher, Waltham, MA USA). A linear gradient elution using solvents 0.1% TFA/H₂O (A) and 0.1% TFA/ACN (B) was carried out as described in Table 1. The fluorescent detector was set to excite at 390 nm and detect emission at 475 nm. Quantitation was conducted by establishing a calibration curve by derivatizing standardized H₂S solutions.

3-aminophthalhydrazide (luminol), 1-(p-hydroxyphenyl)imidazole (HPI), disodium sulfide (Na₂S), erastin, RSL3, cumene hydroperoxide (CHP), liproxstatin-1, 7-(diethylamino)coumarin-3-carbohydrazide (CHH), 4-hydroxytamoxifen (Tam), monobromobimane (MBB), cysteine (Cys), 3-mercaptopyruvate (3MP), cytochrome c (cyt c), glutathione reductase (GR), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), S-nitrosoglutathione (GSNO), ferrocene, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) and isopropyl β-d-1-thiogalactopyranoside (IPTG) were obtained from Sigma-Aldrich. BODIPY C11, diallyl tetrasulfide (DATS) and Amplex Red were obtained from Thermo Fisher Scientific. DEPMPO was obtained from Enzo. H₂O₂ was obtained from Roth. GSH and 5-aminolevulinic acid hydrochloride were obtained from Merck. Sodium disulfide (Na₂S₂), sodium tetrasulfide (Na₂S₄) and 3′,6′-di(O-thiosalicyl)fluorescein (SSP4) were obtained from Dojindo. The synthesis of GSSSG, GS³⁴SSG and GSSSSG was performed as described previously⁴³ . CSSSC was synthesized as described below.