Purpose/Principle

Five different respiratory chain enzyme assays are performed. Citrate synthase and protein are assayed as reference markers. The activity of each enzyme complex is determined by a kinetic spectrophotometric assay over a period of about three to five minutes. Complexes I to IV transfer electrons from one intermediate to the next. In each case, the reduced species absorbs a different amount of light at a particular wavelength. Change in absorbance at 280 nm (coenzyme Q1), 340 nm (NADH) or 550 nm (cytochrome c) or 412 nm (thionitrobenzoate) is used to assay enzyme activity. CoQ1 and decylbenzoquinone (DB) are short-chain analogues of the endogenous hydrophobic CoQ10, which are more suitable for aqueous assay systems. In the assays of complex II, II+III and complex III absorbance increases during the assay, and in other assays absorbance decreases.