Template:SUIT text D084

The coupling-control protocol SUIT-006 NADH mt D084 allows the study of mitochondrial respiration and NADH fluorescence in the three coupling control states LEAK, OXPHOS and ET in the N-pathway.

After the addition of mitochondria in the absence of fuel substrates and ADP, Ren, respiration due to oxidation of endogenous substrates remaining after mitochondrial isolation is measured. If these substrates are fully consumed by the mitochondria, this step can be used for an approximate calibration of oxidized NAD (NAD defined as the sum of the oxidized NAD⁺ and the reduced NADH). If this is not possible, this protocol should be used in combination with SUIT-034 NADH mt D082, where the titration of a small concentration of ADP leads to depletion of endogenous substrates, thus leading to accumulation of oxidized NAD, allowing to calibrate for the fully oxidized NAD.

Anoxia is reached by letting mitochondria fully consume the oxygen in the O2k-chambers. In the absence of O2, the ETS upstream of CIV is reduced and thus leads to an accumulation of reduced NAD. Under anoxia the complex III inhibitor myxothiazol is added and a further increase in the reduced NAD fraction can be observed. This step is then used for the calibration of the fully reduced NAD. At the end of the protocol, the reoxigenation of the chamber allows the measurement of Rox.