Determination of enzymatic activity of mitochondrial Complex IV (cytochrome c oxidase)

As part of our assessment of mitochondrial function, we also measured the enzymatic activity of hepatic mitochondrial Complex IV based on the rationale of Levy et al. [10] who considered that dysfunction of Complex IV was the most important determinant of abnormal mitochondrial function in SS. We determined Complex IV activity spectrophotometrically by a temperature-controlled Ultrospec 2100 ultraviolet-visible spectrophotometer (Biopharmacia Biotech, Uppsala, Sweden) [17]. Complex IV activity was measured at 25 °C by monitoring the absorbance decrease of reduced cytochrome c at 550 nm. The reaction was started by addition of 40 μmol/l reduced cytochrome c into 50 mmol/l phosphate buffer containing 2.5 μg mitochondrial protein solubilized with 0.02 % laurylmaltoside.