Measurement of mitochondrial respiratory chain complex activities

Mitochondria were isolated as previously described⁴³. Briefly, mouse lungs or human PBMCs were homogenized in isolation buffer (1 mM EGTA, 215 mM mannitol, 75 mM sucrose, 0.1% BSA, 20 mM HEPES, pH 7.2). Mitochondria were isolated by differential centrifugation, and homogenized. Mitochondrial respiratory chain complexes I and III enzyme activities were measured using respective assay kits (Nanjing Jiancheng Bio-engineering Institute, Nanjing, China). To measure complex I activity, 900 μl reaction Mix containing ubiquinone and reduced nicotinamide adenine dinucleotide (NADH) was incubated at 30 °C for 3 minutes before 100 μl sample was added. Absorbance at 340 nm (A340) was recorded immediately after the addition of sample and after further incubation for 3 minutes to monitor the rate of NAD production. Absorbance at 380 nm (A380) was also recorded at the two time points for correcting background absorbance. Total complex I activity was calculated based on rate of changes in the corrected A340 readings (A340-A380) and the NAD standard curve. A parallel reaction, in which a specific complex I inhibitor was added into the reaction Mix, was performed for each sample to determine non-specific complex I activity. Specific complex I activity for each sample was calculated by subtracting the non-specific activity from total complex I activity.

To measure complex III activity, 900 μl reaction Mix containing ubiquinol and 2-ferricytochrome c was incubated at room temperature in dark for 10 minutes before 100 μl sample was added. Absorbance at 550 nm (A550) was recorded immediately after the addition of sample and after further incubation for 5 minutes to monitor the rate of reduced cytochrome c production. Complex III activity was calculated based on changes in A550 readings and the reduced cytochrome c standard curve.