Milli q water purification system

Bovine liver catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6, 2000–5000 units mg⁻¹), xanthine oxidase (XO) from bovine milk (xanthine:oxygen oxidoreductase, EC 1.17.3.2, ≥0.4 units mg⁻¹), peroxidase from horseradish (type VI, 310 units mg⁻¹) (HRP), cytochrome c from equine heart (λ_max^{reduced form} = 550 nm), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), superoxide dismutase (1854 units mg⁻¹) (SOD), acetone, 2-isopropanol, manganese(II) chloride tetrahydrate, and other analytical grade chemicals were purchased from Merck KGaA, Darmstadt, Deutschland. Phosphate buffers were prepared from sodium dihydrogen phosphate and disodium hydrogen phosphate. pH 2.0 and pH 3.0 buffers were prepared from phosphoric acid and sodium dihydrogen phosphate. The pH of the solutions was measured using a pH meter (Knick GmbH, Berlin, Germany). The O₂ concentration was monitored using an oxygen sensor (LE621-IP67, MettlerToledo GmbH, Gießen, Germany).
All solutions were prepared using a Milli-Q water purification system (Merk KGaA, Darmstadt, Germany). Before the experiments began, the value of the extinction coefficient, ε₃₄₀, of the ABTS product was examined, and it corresponded to 3.7 × 10⁴ M⁻¹ cm⁻¹ [43 (link)].

Deoxycorticosterone-acetate (DOCA, D7000, Sigma-Aldrich, Sydney, Australia), N,N-dimethylformamide (DMF, ≥99%, Sigma-Aldrich, Sydney, Australia), dimethyl sulfoxide (DMSO, ≥99%, Sigma-Aldrich, Sydney, Australia), noradrenaline (99%,), acetylcholine (≥99%, TLC, Sydney, Australia), sodium nitroprusside (≥99%) and (−)-epicatechin (≥90%, HPLC, Sigma) were purchased through Sigma-Aldrich (Sydney, Australia). With the exception of DOCA, which was dissolved in DMF, all other compounds were dissolved in purified water (Milli-Q water purification system, Merk Millipore, Australia). A stock solution of (−)-epicatechin (1 mg/mL) was made up by dissolving 10 mg (−)-epicatechin in a solution of 9.9 mL purified water and 100 μL DMSO.

Tobramycin (Tob, molecular weight (Mw) 467.5 Da), ciprofloxacin (Cipro, Mw 331.3 Da), itaconic acid (IA, Mw 130.1 Da), peptone, Tween20 and casein were obtained from Merck. Salts and organic compounds of analytical grade (NH₄Cl, KCl, tris-HCl, chloroform, glycerol, glucose, formic acid and MgSO₄·7H₂O) were obtained from Merck and VWR. Yeast extract was obtained from Fluka. Bacto™ Tryptone were obtained from BD Biosciences. Luria Bertani (LB) agar was obtained from Carl Roth. Gibco^® PBS was obtained from Life Technologies. Purified water was produced by Milli-Q water purification system (Merk Millipore, Billerica, MA, USA)
The Pseudomonas aeruginosa strain PA14 wild type (wt) was obtained from ATCC or DSMZ, the German Collection of Microorganism and Cell Cultures GmbH (PA14 = DSMZ19882), stored in glycerol stocks at −80 °C. Bacteria were cultured in a minimal proteose peptone glucose ammonium salt (PPGAS) medium. PPGAS was composed of 1 g/L NH₄Cl, 1.5 g/L KCl, 19 g/L Tris-HCl, 10 g/L peptone, 5 g/L glucose and 0.1 g/L MgSO4·7H₂O. The medium was adjusted to pH 7.2 ± 0.2 and sterilized before use. Agar solution was composed of 15.5 g/L LB agar, 10 g/L peptone, 5 g/L NaCl and 5 g/L yeast extract, the solution was sterilized before being plated.

Water was deionized and passed through a Milli-Q water purification system from Merkmillipore, Darmstadt, Germany. Acetonitrile (ULC-MS) and methanol (ULC-MS) were purchased from Biosolve, Valkenswaard, The Netherlands. Ammonium hydroxide (25%), formic acid (98–100%), acetic acid (100%), dimethylsulfoxide (DMSO), n-hexane, ouabain and veratridine were purchased from Merck, Darmstadt, Germany. Certified Tetrodotoxin (TTX) material (25.8 ± 2.1 µg/g) was purchased from Cifga, Lugo, Spain. Non-certified TTX material was purchased from Latoxan, Portes lès Valence, France.

The following materials used in this study were purchased from Sigma-Aldrich (Johannesburg, South Africa): Al powder (<200 mm, 95% purity), Bi granules (>99.9% purity), and Zn powder (<150 mm, >99.8% purity). Hydrolysis procedures were performed using tap water, filtered water, and deionized water (18.2 MΩ cm⁻¹ resistivity) produced by a Milli-Q water purification system (Sigma-Aldrich, Johannesburg, South Africa). Some hydrolysis reactions were performed in sodium chloride (NaCl) solutions. The NaCl (99.5%) was supplied by Sigma-Aldrich (Johannesburg, South Africa). Pure nitrogen gas (99.9%; Afrox, Johannesburg, South Africa) was used for all purging procedures.