An Experimental Approach to Address the Functional Relationship between Antioxidant Enzymes and Mitochondrial Respiratory Complexes

Abstract

Mitochondrial dysfunction and cytosolic oxidative stress are pathological biomarkers interlinked in several chronic diseases and cellular toxicity promoted by high-energy radiation or xenobiotics. Thus, assessing the activities of the mitochondrial redox chain complexes and the cytosolic antioxidant enzymes in the same cell culture system is a valuable approach to addressing the challenge of chronic diseases or unveiling the molecular mechanisms underlying the toxicity of physical and chemical stress agents. The present article gathers the experimental procedures to obtain, from isolated cells, a mitochondria-free cytosolic fraction and a mitochondria-rich fraction. Furthermore, we describe the methodologies to evaluate the activity of the main antioxidant enzymes in the mitochondria-free cytosolic fraction (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase), and the activity of the individual mitochondrial complexes I, II and IV, as well as the conjugated activity of complexes I–III and complexes II–III in the mitochondria-rich fraction. The protocol to test the citrate synthase activity was also considered and used to normalize complexes. The procedures were optimized within an experimental setup to allow that each condition to be tested only requires sampling of one T-25 flask of cells 2D cultured, as the typical results presented and discussed here.