MitoCheck® Complex II/III Activity Assay Kit Reference: 700950-96 Complex III (CoQ cytochrome c oxidoreductase) is an essential protein for mitochondrial oxidative phosphorylation. Complex III functions as both a gatekeeper for mitochondrial respiration and as a major source of reactive oxygen species III. It accepts electrons from complexes I and II in the form of QH2, the reduced form of the electron carrier ubiquinone. Once bound to complex III, QH2 undergoes a series of redox reactions, known as the Q-cycle. During the Q-cycle, electrons are passed from QH2 onto cytochrome c via the Rieske iron-sulfur protein and cytochrome c1 resulting in the translocation of 4H+ and the generation of 02•-. Cayman's MitoCheck® Complex II/III Activity Assay measures the reduction of excess cytochrome c (550 nm absorbance) as catalyzed by complex III. This assay is coupled to succinate co-enzyme Q oxidoreductase (complex II) for the generation of QH2. Due to the dependence on complex II activity, a counter-screen for complex II activity (Item No. 700940) should be performed in order to truly measure complex III activity.
MitoCheck® Complex IV Activity Assay Kit Reference: 700990-96 Complex IV (cytochrome c oxidase) is the terminal electron acceptor in the mitochondrial electron transport chain. Complex IV functions by oxidizing cytochrome c and completing a four-electron reduction of oxygen (O2) to form water. During this process, two H+ are translocated from the mitochondrial matrix to the intermembrane space, contributing the mitochondrial membrane potential required for ATP synthesis. Common inhibitors of complex IV include potassium cyanide, azide, O2 limitation, nitric oxide, and carbon monoxide. Cayman’s MitoCheck® Complex IV Activity Assay is designed to measure the direct oxidation of cytochrome c by complex IV, in an isolated bovine heart mitochondrial system that is supplied within the kit.
MitoCheck® Complex V Activity Assay Kit Reference: 701000-96 The mitochondrial F1FO ATP synthase, commonly referred to as complex V, is a multi-subunit protein that utilizes energy in the form of a proton gradient generated by the electron transport chain, to phosphorylate ADP to form ATP in the presence of Pi. This mechanism, referred to as oxidative phosphorylation, is responsible for ~90% of ATP generation in mammalian cells. Cayman's MitoCheck® Complex V Activity Assay measures the activity of Complex V, in the reverse reaction, as an ATPase in the mitochondrial samples provided. In this assay, ATP is dephosphorylated ADP by complex V. The ADP is then utilized by pyruvate kinase to convert phosphoenolpyruvate into pyruvate with the concomitant generation of ATP. Pyruvate, in the presence of NADH and lactate dehydrogenase, is then reduced to lactate and NAD+. The rate of NADH oxidation is monitored at 340 nm. Isolated bovine heart mitochondria are provided in this kit. Tissue mitochondria can also be acquired using Cayman’s MitoCheck® Mitochondrial (Tissue) Isolation Kit (Item No. 701010).