Glutathione peroxidase

Sample (20 µL, 5 mg/mL of pure DMSO) was mixed with 8 μL of EDTA solution, 10 μL of glutathione reductase (0.2 U Sigma G3664), 4 μL of GSH solution, 10 μL of glutathione peroxidase (0.04 U Sigma G6137), 22 μL of H₂O₂ and 332 μL of 50 mM sodium phosphate, pH 7.0). To start the reaction, 4 μL of NADPH solution (N5130) was added and the decrease in the absorbance (340 nm) was read after 10 min of incubation at 25 °C. All solutions were prepared in 50 mM buffer and concentrations of reagents in the final mixture were as follows: 1mM EDTA, 0.2 U glutathione reductase, 2 mM GSH, 0.04 U glutathione peroxidase, 1.5 mM H₂O₂ and 0.8 mM NADPH. Blank sample was prepared with buffer instead of the studied sample and background was measured (mixture containing studied sample and buffer only). One unit of enzyme activity was defined as nMol of NADPH consumed/min·mL sample, in comparison with nMol of NADPH consumed/min in blank (reagent) sample [76 (link)].

Thiobarbituric acid (TBA), sodium dodecyl sulfate (SDS), glacial acetic acid, N-butanol, pyridine, 1,3,3-tetraethoxypropane (TEP), cytochrome C, xanthine oxidase, xanthine, glutathione reductase, nicotinamide adenine dinucleotide phosphate (NADPH), hydrogen peroxide, superoxide dismutase, glutathione peroxidase, catalase, acetylthiocholine iodide (ATCI), acetylcholinesterase, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), cresyl violet, sodium acetate, sodium carbonate, 2,4,6-tripyridyl-striazine (TPTZ), Folin-Ciocalteu reagent, gallic acid, ascorbic acid, Trizma hydrochloride, potassium chloride, 2,2-diphenyl-1-picrylhydrazyl (DPPH), tris-hydrochloride, and sodium carbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemicals used in Western blot analysis were purchased from Bio-Rad Laboratories. Methanol and acetic acid (HPLC grade) were purchased from Fisher Scientific.

The following reagents were purchased from Sigma Aldrich (St. Louis, MO, USA): 7-hydroxy-6-methoxycoumarin (scopoletin), curcumin, p-dimethylaminobenzaldehyde, 1,1,3,3-tetraethoxypropane (TEP), chloramines-T, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), reduced glutathione, glutathione reductase (GSH-Rd), glutathione peroxidase (GSH-Px), β-nicotinamide adenine dinucleotide phosphate (β-NADP), and β-NADPH. Perchloric acid was obtained from GFS Chemical Co. (Columbus, OH, USA), thiobarbituric acid (TBA) from Lancaster Co. (Lancashire, England, UK), and hydrogen peroxide from Junsei Chemical Co., Ltd. (Tokyo, Japan).

1,1-diphenyl-2-picryhydrazyl (DPPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT), 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA), 4’,6-diamidino-2- phenylindole dihydrochloride (DAPI), 6-hydroxy-2,5,7,8-tetramethy-chroman-2-carboxylic acid (trolox), 6-hydroxydopamine bromide (6-OHDA), 2’,7’-dichlorofluorescein diacetate (DCFH-DA), acridine orange (AO), ascorbic acid, (+)-catechin, caffeic acid, dimethyl sulfoxide (DMSO), epicatechin, ferrous sulfate heptahydrate, Folic-Ciocalteu’s phenol reagent (FCP), gallic acid, glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), hydrogen chloride, malodialdehyde (MDA), mangiferin, phosphate buffered saline (PBS), quercetin, sodium molybdate, sodium nitrate, superoxide dismutase (SOD), thiobarbituric acid (TBA), trichloroacetic acid (TCA), vanillic acid, verbascoside, xanthine, and xanthine oxidase (XO) were obtained from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). Hydrogen peroxide (H₂O₂) and all HPLC-grade solvents were acquired from Merck (Darmstadt, Germany). Dulbecco’s modified Eagles medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Double distilled water was used throughout the experiments.

Glutathione peroxidase (GPx, EC 1.11.1.9) activity was evaluated by the decrease of NADPH (Sigma Chemical Co., St. Louis, MO, USA) concentration at 340 nm. The reaction medium contained 100 mM potassium phosphate buffer (Sigma Chemical Co., St. Louis, MO, USA), pH 7.7, 1 mM EDTA (Sigma Chemical Co., St. Louis, MO, USA), 2 mM reduced glutathione (Sigma Chemical Co., St. Louis, MO, USA), 0.15 U/mL glutathione reductase (Sigma Chemical Co., St. Louis, MO, USA), 0.4 mM azide (Sigma Chemical Co., St. Louis, MO, USA), 0.1 mM NADPH, and 0.5 mM tert-butyl hydroperoxide (Sigma Chemical Co., St. Louis, MO, USA) as enzyme substrate. GPx unit is defined as 1 µmol of NADPH consumed per minute and the specific activity as units/mg protein [42 (link)].

GSH and GSSG levels were measured as previously described (21 (link)). GSH and GSSG levels were determined as the difference in low molecular weight thiols. BAL fluid, cell lysates, or apical culture supernatants were incubated with glutathione peroxidase (Sigma-Aldrich, G6137), cumene hydroperoxide (Sigma-Aldrich, 247502), glutathione reductase (Sigma-Aldrich, G9297), and NADPH (Sigma-Aldrich, N1630) for 30 minutes at 25°C. Thiol Fluorescent Probe IV (10 mM; Sigma-Aldrich, 1173888-41-9) was then used to identify the low molecular weight thiols, with fluorescence measured at 400 nm excitation and 465 nm emission. A standard curve was established by the addition of GSH to PBS. Intracellular GSH and GSSG levels were corrected for intracellular protein, determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) using albumin as a standard. The lower limit of detection for this assay was 0.016 μM or 0.13 nmol/mg protein.