NADH Dehydrogenase Activity Assay

NADH dehydrogenase activity was measured in 1 mL NDH buffer (50 mm potassium phosphate buffer, pH 7.5, 1 mm EDTA, 0.2 mm KCN), containing 20–30 μg proteins from the mitochondrial lysates and 5 μL of 20 mm NADH (AppliChem). After the addition of 2 μL 10 mm coenzyme Q₂ (Sigma-Aldrich), the change in absorbance at 340 nm was followed for 3 min (Čermáková et al., 2019 (link)). A unit of activity was defined as the amount of enzyme that catalyses the oxidation of 1 nmol NADH per min, assuming an extinction coefficient of 6.2 L mmol⁻¹ cm⁻¹ (Gonzalez-Halphen and Maslov, 2004 (link)). Solutions of the inhibitors were freshly prepared. Capsaicin (Sigma-Aldrich) was dissolved in ethanol, rotenone (Serva, Heidelberg, Germany) and DPI (diphenyl iodonium, Sigma-Aldrich) – in dimethylsulphoxide and methanol, respectively. Rotenone and DPI were added to the assay mixture immediately before the start of the reaction, Capsaicin was pre-incubated for 3 min. Native electrophoresis and in-gel activity staining methods were adapted from Zerbetto et al. (1997 (link)) and Wittig et al. (2007 (link)) and performed as described previously (Verner et al., 2014 (link)).