Dibromobimane

H₂S release from GluSH was measured in ddH₂O at 37 °C over 7 days. GluSH loaded into a Transwell membrane insert (Corning) was placed in centrifuge tubes containing Hank’s buffer saline solution (HBSS) supplemented with excess amounts of bicarbonate (1.5 mol per 1 mol GluSH). Each centrifuge tube was sealed with vacuum glue and parafilm. At certain time points, 1 mL of the release solution was extracted from the reaction tube using a needle and replaced with 1 mL of HBSS and bicarbonate solution. The solutions were then immediately assayed to determine their H₂S concentration using a fluorescent method measuring the conversion of monobromobimane (MBB) to thiobimane [41 (link)]. Briefly, dibromobimane (Sigma-Aldrich) was dissolved in HBSS at a concentration of 500 µM and incubated with the test samples at room temperature for 5 min after which florescence (ex. 340 nm, em. 465 nm) was measured. A standard curve was created by dissolving different concentrations of NaSH in the 500 µM dibromobimane in HBSS solution, incubating at room temperature for 5 min, and measuring the florescence. The concentration of the H₂S at each time point was then calculated by comparing the test sample values to the NaSH standard curve.

Dimethylsulfoxide (DMSO), monosodium and disodium phosphates, Tris, Pipes, DTT, PMSF, Zincon, diamide, and dibromobimane (DBB, spacer arm, 4.88 Å [50 (link)]) were from Sigma. Tris(2-maelimidoethyl)amine, (TMEA, spacer arm, 10.3 Å) and bismaleimidohexane (BMH, spacer arm, 13.0 Å) were from Thermo Fisher Scientific. Monobromobimane (MBB, sc-214629) and (+)-Biotin-(PEO)3-iodoacetamide (biotinylated iodoacetamide, Iac-B) were purchased from Santa Cruz Biotechnology. Citric acid, sodium carbonate and bicarbonate, 2-mercaptoethanol, and EDTA were obtained from Merck. Precision Plus-Dual Color protein standards and Sypro Ruby were from BioRad. The biotinylated analog of 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂-B) was from Cayman Chemical. No unexpected or unusually high safety hazards were encountered throughout this work.

For crosslinking experiments, 5 µM SUMO-PglC in PglC buffer (50 mM HEPES, 100 mM NaCl, 5% glycerol, 0.2% DDM, pH 7.5) was treated with 25 µM dibromobimane (Sigma-Aldrich, St. Louis, MO) from a 1 mM stock in 20% acetonitrile for 20 min in the dark at room temperature. For fluorescence analysis, some crosslinked samples were further treated with 50 mM DTT for 1 min, and all samples were then analyzed by SDS-PAGE and quantified by gel densitometry using a Molecular Imager Gel Doc XR+ System with Image Lab software (BioRad). Fluorescence was measured using the ethidium bromide excitation setting (302 nm) and normalized to band intensity after Coomassie staining. Relative fluorescence of SUMO-PglC variants is reported as fluorescence intensity, normalized to Coomassie staining, relative to DTT-quenched samples. Technical replicates were performed on distinct aliquots taken from a common protein purification prep.
For activity assays crosslinked SUMO-PglC prepared as above was diluted with PglC buffer to a 50 nM stock and assayed as described below. Activity of variants is reported relative to control samples that were treated with carrier (20% acetonitrile) and assayed in parallel with crosslinked samples. Data were plotted using Graphpad Prism 7 (GraphPad Software, RRID:SCR_002798).