Biochemical Assays of Respiratory Chain Complex Activity

Abstract

Publisher Summary This chapter focuses on the biochemical assays of the respiratory chain (RC) complex activity. It presents the preparation of mitochondrial fractions from tissues and cultured cells for RC enzymology; the measurement of activity of the individual complexes I, II, III, IV, V, the mitochondrial matrix marker enzyme citrate synthase; and the combined activity of complexes II + III. RC enzyme activities are frequently expressed relative to its activity of citrate synthase. Such ratios are more robust than absolute activities because of the variability inherent in cell culture conditions, such as passage number and the degree of confluence, and the proliferation of mitochondria seen in tissues of many patients with mitochondrial disease. The effects of postmortem delay on RC enzymes from liver samples frozen at varying times after death were analyzed to assess the stability of RC enzyme activities postmortem. There can be considerable loss of RC enzyme activity postmortem, particularly in liver, but the observations suggest that muscle collected and frozen at -70°C within 6 h of death and liver within 2 h remain suitable for RC enzyme analysis. The chapter presents the effects of tissue pathology by comparing RC enzymes in tissues from patients without RC complexes I–IV defects with normal controls. The possibility of secondary decreases in enzyme activity and the broadening of reference ranges in the presence of tissue pathology should be considered in interpreting RC enzyme profiles.