How many molecules of mitochondrial complex I are in a cell?

Mitochondrial complex I is the only enzyme responsible for oxidation of matrix NADH and regeneration of NAD⁺ for catabolism. Nuclear and mtDNA mutations, assembly impairments, and enzyme damage are implicated in inherited diseases, ischemia-reperfusion injury, neurodegeneration, and tumorogenesis. Here we introduce a novel method to measure the absolute content of complex I. The method is based on flavin fluorescence scanning of a polyacrylamide gel after separation of complexes by Clear Native electrophoresis. Using mouse primary astrocytes as an example, we calculated an average value of 2.2 × 10⁵ complex I molecules/cell. Our method can be used for accurate quantification of complex I content.