Mitochondrial electron transport chain (ETC) complexes activity assays

Mitochondrial respiratory chain enzymatic activities (complexes I-IV) were assessed as previously described ¹⁴ with slight modification:

Complex I activity: Prepared mitochondria (2 µl; 2 μg/µl) were added to the assay medium and the baseline was read at 340 nm for 2 minutes. Ubiquinone 1 (1µl; 5 mM) was applied and the decrease in absorbance was recorded at 340 nm for 2 minutes. In parallel, the same quantity of reagents and samples but with the addition of rotenone solution (1µl; 1 mM) was used.

Complex II activity: Prepared mitochondria (2 µl; 2 μg/µl) were added to the assay medium and the mixture was incubated at 37°C for 8 minutes and then the baseline was recorded at 600 nm for 2 minutes. Then DUB (1µl;5 mM) was applied and the decrease in absorbance at 600 nm was recorded for 2 minutes. In parallel, malonate (1µl; 1 M) was used.

Complex III activity: Prepared mitochondria (2µl; 2 μg/μL) were added to the assay medium. After reading the baseline at 550 nm for 2 minutes, decylubiquinol (1µl;5 mM) was used and then the increase in absorbance at 550 nm was recorded for 2 minutes. In parallel, the same quantity of reagents and samples with the addition of antimycin A (1µl; 1 mg/ml) were used.

Complex IV activity: Reduced cytochrome c (5 µl; 1 mM) was added to the assay medium. The baseline activity was recorded at 550 nm for 2 minutes. Then the prepared mitochondria (2 µl; 2 μg/µl) were added and the decrease in absorbance at 550 nm was recorded for 2 minutes. The specificity of complex IV activity was checked in parallel experiment, in which NaN3(1µl; 0.5 M) was added. The enzymatic activities for each mitochondrial enzyme should be calculated according to the following equation: Enzyme activity (nmol min^-1 mg^-1) = (∆Absorbance/min × 1,000)/[(extinction coefficient × volume of sample used in ml) × (sample protein concentration in mg ml^-1)].