All activities were measured spectrophotometrically using Molecular Devices SpectraMax M2 plate reader in 0.2 ml of assay buffer composed of 125 mM KCl, 20 mM Hepes–KOH (pH 7.5), 0.02 mM EGTA, at 25 °C.
NADH-dependent activities of complex I were assayed as oxidation of 100 μM NADH at 340 nm (ε340nm = 6.22 mM−1 cm−1) in the assay buffer supplemented with either 1 mM HAR or 40 to 50 μM Q1. Mouse brain mitochondria activities were measured using 10 to 30 μg/ml for HAR and 100 to 125 μg/ml of DDM-solubilized brain mitochondria for Q reductase activities. For tissue homogenates, DDM-solubilized protein was added to concentration of 2 to 30 or 50 to 400 μg/ml for HAR and Q1 reductase activities, respectively.
NADH-dependent activities of complex I were assayed as oxidation of 100 μM NADH at 340 nm (ε340nm = 6.22 mM−1 cm−1) in the assay buffer supplemented with either 1 mM HAR or 40 to 50 μM Q1. Mouse brain mitochondria activities were measured using 10 to 30 μg/ml for HAR and 100 to 125 μg/ml of DDM-solubilized brain mitochondria for Q reductase activities. For tissue homogenates, DDM-solubilized protein was added to concentration of 2 to 30 or 50 to 400 μg/ml for HAR and Q1 reductase activities, respectively.