Optimal conditions for determination of cytochrome c oxidase activity in the rat heart

The determination of cytochrome c oxidase (COX) activity represents an impo rtant indicator for the evaluation of cell oxidative capacity. However, it has been shown repeatedly that different factors modify the rate of COX act ivity under various experimental conditions. The most important concern the ionic concentrations of the medium and the application of various detergen ts for the solubilization of mitochondrial membranes. We found the highest activity of COX in rat heart homogenates and mitochondria at 40-60 mM potas sium phosphate. The rate of COX activity is dependent on the detergent/prot ein (P) ratio. Using n-dodecyl-beta-D-maltoside (lauryl maltoside, LM) as t he detergent, we obtained the highest activity at LM/P ratios of(50: 100):1 . By kinetic measurements of low-affinity binding sites in heart mitochondr ia, we found V-lim values of 4.3 and 22.2 mu mol cytochrome c per min per m g P in the presence or absence of lauryl maltoside, respectively. The K-m v alues were 16.7 mu mol in the presence or absence of lauryl maltoside. Our results thus indicate that 1) the exact assessment of COX activity in heart homogenates and mitochondria requires the determination of optimum phospha te concentrations in the medium used, and 2) even small modifications of th e experimental procedure may induce significant differences in the maximum values of COX activity.