Ferric nitrate reagent

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.

The activity of TST was determined using Sörbo’s method [49 ] following a procedure used by Wróbel and others [48 (link)]. The reaction mixture consisted of 200 µL of 0.125 M sodium thiosulfate (Sigma-Aldrich, Darmstadt, Germany), 100 µL of 0.2 M potassium dihydrogen phosphate (Sigma-Aldrich, Darmstadt, Germany), 100 µL of homogenates, 100 µL of 38% formaldehyde (only blank sample; Polish Chemicals Reagents, Gliwice, Poland), and 100 µL of 0.25 M potassium cyanide (KCN, Sigma-Aldrich, Darmstadt, Germany). After 5 min incubation at room temperature, the following reagents were added: 100 µL of 38% formaldehyde (only to blank samples) and 500 µL of 0.2 M ferric nitrate reagent (Sigma-Aldrich, Darmstadt, Germany). The amount of thiocyanate formed during the reaction catalyzed with TST was measured colorimetrically at 460 nm. The enzyme units were defined as nmoles of SCN⁻ (thiocyanate), which formed during 1 min incubation per 1 mg of protein.

TST activity was assayed by the Sörbo’s method [42 ], following a procedure described by Wróbel and others [43 (link)]. The incubation mixture contained: 200 μL of 0.125 M sodium thiosulfate (Sigma-Aldrich, Poznan, Poland), 100 μL of 0.2 M potassium dihydrogen phosphate (Sigma-Aldrich, Poznan, Poland), 100 μL of homogenates, 100 μL of 38% formaldehyde (Avantor Performance Materials Poland S.A., Gliwice, Poland) (only blank sample) and 100 μL of 0.25 M potassium cyanide (Merck, Darmstadt, Germany). The mixture was incubated for 5 min at room temperature. Subsequently, 100 μL of 38% formaldehyde (only tested sample) and 500 μL of 0.2 M ferric nitrate reagent (Sigma-Aldrich, Poznan, Poland) were added to all samples. The amount of thiocyanate formed during the reaction catalyzed by TST was measured colorimetrically at 460 nm (Genesys 10UV Scanning UV/Visible Spectrophotometer, Thermo Scientific, Waltham, MA, USA). The enzyme units were defined as nmoles of SCN⁻, which are formed during 1 min incubation per 1 mg protein.