Glutathione disulfide

Fast Blue BB salt, hypoxanthine, Triton X-100, thiobarbituric acid, trichloroacetic acid, dimethyl sulfoxide (DMSO), bovine serum albumin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl bromide (MTT), phosphate-buffered saline (PBS), D-sorbitol, acridine orange, ethidium bromide, buthionine sulfoximine (BSO), glutathione disulfide, glutathione reductase, o-phthalaldehyde, nicotinamide adenine dinucleotide phosphate (NADPH), chloroquine diphosphate, artesunate, and clotrimazole were purchased form Sigma-Aldrich (St. Louis, MO, United States), and Albumax II, Roswell Park Memorial Institute 1,640 (RPMI-1640), Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), and fungizone were purchased from Gibco (Grand Island, United States). Griess reagent kit, chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), and SYBR Green qPCR super mix were purchased from Invitrogen (Carlsbad, CA, United States). MitoScreen Flow Cytometry kit (Cat No.551302) and APO-BrdU apoptosis detection kit (Cat No. 556381) were obtained from BD Biosciences (Franklin Lakes, NJ, United States).

HPLC was performed on a Restek C₁₈ 5-μm reverse phase column (250 × 4.6 mm; Restek Corp., Bellefonte, PA). The running buffer was 10 mm KPO₄ buffer, 10 mm tetrabutylammonium bisulfate, 5% acetonitrile, pH 7.0 at a flow rate of 1 ml/min. Samples were run using a Shimadzu HPLC equipped with Class VP V. 7.4 software (Shimadzu, Kyoto, Japan). Elution times and UV-visible spectra were compared with authentic standards of nitrite, GSNO, glutathione disulfide, and GSH (Sigma) prepared and run using the same elution buffer.
All solutions of protein and salts were prepared using argon-purged buffer. All samples were prepared in an oxygen-free glove box. A stock solution of metLtHb (0.3 mm in heme) in 0.05 m Bis-Tris, pH 7.0 was prepared and divided into two samples of equivalent volume. One-half was treated with both NONOates and nitrite to produce the NA intermediate, and the second sample was treated with NONOates alone and allowed to evolve into the ferrous NO derivative.

Reduced form of L-glutathione was obtained from KOHJIN Life Sciences (Tokyo, Japan). Glutathione disulfide, γ-L-glutamyl-L-Cysteine, L-cysteinyl-glycine, and L-cystine with ¹³C isotopes at C1 and C1′ (L-Cystine-1,1′-¹³C) were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-Cysteine was obtained from Wako Pure Chemicals (Osaka, Japan), 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ) from Synchem (Altenburg, Germany), and the AccQ. Tag Ultra Derivatization Kit from Waters (Milford, MA, USA). Acetonitrile (HPLC-grade), triethylamine, formic acid, trichloroacetic acid (TCA), 2-mercaptoethanol, and N-ethylmaleimide (NEM) were obtained from Nacalai Tesque (Kyoto, Japan). Amino acid derivatives and reagents for peptide synthesis were purchased from Watanabe Chemical (Hiroshima, Japan).

Glutathione, Glutathione disulfide (GSSG), antibiotics (ciprofloxacin, amikacin, Augmentin, gentamicin and tobramycin), resazurin dye, crystal violet solution, Phosphate buffered saline (1 × PBS) and Hydrogen peroxide (H₂O₂) were all obtained from Sigma-Aldrich (Sydney, Australia). Tryptone Soy Broth (TSB) was obtained from Oxoid (Thermo Fisher Scientific, Australia), Enzymes: DNase-I was obtained from Invitrogen (Melbourne, Australia), α-Amylase from MP Biomedicals (NSW, Australia) and Proteinase K from Sigma-Aldrich (Sydney, Australia). Dulbecco’s Modified Eagle Medium (DMEM) and Fetal bovine serum (FBS) from Sigma-Aldrich (Sydney, Australia).