Development of a New Assay for Complex I of the Respiratory Chain

Abstract

AbstractBackground: Measurement of complex I activity has been hampered by the large amounts of tissue required and the resulting turbidity of the assay solution, which makes spectrophotometric analysis difficult. We have developed a new assay for measuring the activity of complex I in isolated mitochondria that is also applicable to skeletal muscle homogenate in patients with suspected mitochondrial diseases.Methods: The method was a radioenzymatic assay based on the preferential oxidation of the 4B hydrogen of NADH by complex I. We prepared tritiated isoforms of NADH for both the respective 4A-3H and 4B-3H positions. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or human skeletal muscle and incubated with the respective radioisotopes. The product (3H2O) was collected after charcoal adsorption of unreacted NADH and taken as an indicator of NADH oxidation. Sensitivity to rotenone was used as a measure of complex I specific activity.Results: The assay was linear with time and protein for isolated mitochondria and tissue homogenates from rats and humans. The Vmax and Km values obtained for 4B-NADH with isolated rat skeletal muscle mitochondria were 35 μmol/L and 90 μmol · min−1 · mg protein−1, respectively. The assay was reproducible and useable for routine measurements in human skeletal muscle. The sensitivity was >10-fold higher than the sensitivities of spectrophotometric techniques.Conclusions: The results of our studies demonstrate the successful development of a new assay for complex I that is rapid, easy to perform, and that enables the processing of multiple samples at one time.