Colorimetric Assay of Complex I Activity

Complex I (NADH:ubiquinone oxidoreductase) activity was measured using mitochondrial Complex I Activity Colorimetric Assay Kit (Bio-vision, Milpitas, California, USA), according to Ansari et al. [33 (link)]. The kit uses decylubiquinone, an analog of ubiquinone, as an electron acceptor that gets converted to decylubiquinol through the catalytic activity of complex I. The complex I dye absorbs light at 600 nm in its oxidized form and accepts electrons from decylubiquinol. Complex I activity was determined colorimetrically by recording the change in absorbance of reduced complex I dye at 600 nm. Complex I activity was calculated from the equation:
$$\mathrm{C}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{x}\mathrm{I}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{m}\mathrm{U}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{s}/\mathrm{\mu }\mathrm{g})\frac{\mathrm{\Delta }\left[\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{x}\mathrm{I}\mathrm{d}\mathrm{y}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right]}{\mathrm{\Delta }\mathrm{t}\mathrm{x}\mathrm{p}\mathrm{x}\mathrm{D}}$$
where Δ [reduced complex I dye concentration] is the change in reduced complex I dye concentration during Δt, Δt = t₂ –t₁ (min), p is the mitochondrial protein (μg) and D is the dilution factor.
Then the net complex I activity in the sample was calculated by subtracting the activity in reaction without rotenone minus the activity in reaction with rotenone. One unit of complex I is the amount of enzyme that causes the reduction of 1 μmol of the dye per min at pH 7.4 at room temperature.