Table 3.
Description of spectrophotometric enzymatic assays to measure respiratory complex activity
| Respiratory complex (synonyms) | Enzymatic assay description |
|---|---|
| Complex I (NADH:ubiquinone oxidoreductase, NADH dehydrogenase, EC 1.6.5.3) | In catalysing the oxidation of NADH to NAD+, Complex I transfers electrons from NADH to ubiquinone (coenzyme Q10). Assays to measure Complex I activity therefore utilise NADH as the electron donor, and a ubiquinone analogue (e.g. coenzyme Q1 or decylubiquinone, DB) as the electron acceptor. Activity is determined by measuring the rate of oxidation of NADH to NAD+, or less frequently by measuring the rate of reduction of the ubiquinone analogue. The rate of NADH oxidation is measured by tracking the decrease in absorbance of the sample at 340 nm, or DB reduction can be followed at 247–272 nm. The majority of Complex I activity should be rotenone-sensitive. |
| Complex II (succinate-ubiquinone oxidoreductase, succinate dehydrogenase) | Complex II oxidises succinate to fumarate, while reducing ubiquinone to ubiquinol. In this assay, succinate is used as the electron donor with dichlorophenolindophenol (DCPIP) the electron acceptor. Complex II activity can be measured by following the decrease in absorbance at 600 nm caused by the reduction of DCPIP. Complex II activity should be sensitive to malonate. Rotenone and antimycin A are usually added to minimise the endogenous ubiquinone accepting electrons from Complex II (and the resultant underestimation of Complex II activity). |
| Complex III (ubiquinol-cytochrome c oxidoreductase, EC 1.10.2.2) | In catalysing the reduction of cytochrome c, complex III transfers electrons from ubiquinol to cytochrome c. A ubiquinone analogue (such as DB) is therefore used as the electron donor, while cytochrome c is used as the electron acceptor. Complex III activity is measured by following the increase in absorbance at 550 nm caused by the reduction of cytochrome c. The rate of oxidation of the ubiquinone analogue can also be monitored. The majority of Complex III activity should be sensitive to antimycin A. Cyanide (CN) or another Complex IV inhibitor should be added to prevent cytochrome c oxidation. |
| Combined complex I and complex III (NADH-cytochrome c oxidoreductase) | The rate of reduction of cytochrome c (electron acceptor), using NADH as the electron donor (Complex I substrate), is measured by monitoring the increase in absorbance at 550 nm. CN or another Complex IV inhibitor should be added to prevent cytochrome c oxidation. |
| Combined complex II and III (succinate-cytochrome c oxidoreductase) | The rate of reduction of cytochrome c (electron acceptor), using succinate as the electron donor (Complex II substrate), is measured by tracking the increase in absorbance at 550 nm. CN or another Complex IV inhibitor should be added to prevent cytochrome c oxidation. |
| Complex IV (cytochrome c oxidase, EC 1.9.3.1) | Complex IV transfers electrons from cytochrome c to molecular oxygen, converting oxygen to water. To measure Complex IV activity, reduced cytochrome c (ferrocytochrome c) is used as the electron donor. Complex IV activity is measured by the decrease in absorbance at 550 nm, caused by oxidation of cytochrome c. The majority of complex IV activity is sensitive to potassium cyanide (KCN). |
| F1Fo ATP synthase | The activity of the F1Fo ATP synthase (assayed as the reverse ATPase activity) is more difficult to measure, due to a high level of oligomycin-resistant ATPase activity [57]. Assays can be performed that infer the reverse activity of ATP synthase (ATP hydrolysis to ADP) by measuring the lactate dehydrogenase-driven oxidation of NADH to NAD+. These reactions are coupled by the activity of pyruvate kinase which supplies pyruvate to fuel lactate dehydrogenase while generating ATP to fuel the ATP synthase reverse activity. Enzyme activity is measured by tracking the decrease in absorbance at 340 nm caused by NADH oxidation [43]. |
Protocols for a variety of assays can be found on manufacturers’ websites for commercially available assays (e.g. the MitoToxTM suite from MitoSciences, or MitoCheck® from Cayman), and in various publications (e.g. [43, 57, 94]). Respiratory chain complex activities can be normalised to the amount of protein in the sample, or to citrate synthase activity [43, 57].