Genesys 10uv scanning uv visible spectrophotometer

Paraoxonase activity was determined by measuring the increase in absorbance at 412 nm (Thermo Scientific GENESYS 10 UV Scanning UV/Visible Spectrophotometer) due to the formation of 4-nitrophenol (using paraoxon as substrate) (Zehra et al., 2009). The assay mixture contained 1.0 mM paraoxon and 1.0 mM CaCl₂ in 50 mM glycine/NaOH buffer (pH 10.5). The amount of 4-nitrophenol liberated was calculated from the molar coefficient 18,290/Mcm.

Sulfane sulfur level was determined by the method of Wood [47 (link)]. This method is based on a cyanolysis reaction and colorimetric determination of ferric thiocyanate complex ion. Incubation mixtures in a final volume 880 μL contained: 20 μL 1 M ammonia solution (Avantor Performance Materials Poland S.A., Gliwice, Poland), 20 μL homogenate, 740 μL H₂O, and 100 μL 0.5 M potassium cyanide (Merck, Darmstadt, Germany). The incubation was performed for 45 min at room temperature. After incubation, thiocyanate was estimated colorimetrically at 460 nm (Genesys 10UV Scanning UV/Visible Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA) after addition of 20 μL 38% formaldehyde (Avantor Performance Materials Poland S.A., Gliwice, Poland) and 40 μL ferric nitrate reagent (Sigma-Aldrich, Poznan, Poland). The level of sulfane sulfur was expressed as nmoles of SCN^- (thiocyanate) per 1 mg of protein.

An MPST activity was assayed according to the method of Valentine and Frankenfeld [44 (link)]. The incubation mixture contained: 250 μL of 0.12 M sodium phosphate buffer, pH 8.0, 50 μL of 0.5 M sodium sulfate (Sigma-Aldrich, Poznan, Poland), 50 μL of 0.15 M D, L-dithiothreitol (DTT, Sigma-Aldrich, Poznan, Poland), 50 μL of distilled water, 50 μL of supernatant and 50 μL of 0.1 M 3-mercaptopyruvate acid sodium salt (Sigma-Aldrich, Poznan, Poland) of the final volume of 500 μL. The mixture was incubated for 15 min, then 250 μL of 1.2 M PCA was added to stop the reaction. Samples were centrifuged at 1600× g for 5 min, and then 100 μL of supernatant was transferred to a 1350 μL solution containing: 1200 μL of 0.12 M sodium phosphate buffer, pH 8.0, 100 μL of 0.1 M N-ethylmaleimide (NEM, Sigma-Aldrich, Poznan, Poland) and 50 μL of β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH, Sigma-Aldrich, Poznan, Poland) (5 mg/mL). After equilibration at 37 °C, 2.5 μL (7 IU) of L-lactate dehydrogenase (LDH, Sigma-Aldrich, Poznan, Poland) was added, and the decrease in absorbance was measured at 340 nm (Genesys 10UV Scanning UV/Visible Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). The MPST activity was expressed as nmoles of pyruvate produced during one minute-incubation at 37 °C per 1 mg of protein.