Optimum uncoupler concentration
Uncoupler titrations
A titration of an uncoupler is necessary to achive the optimum concentration necessary for maximum stimulation of non-coupled respiration (ETS capacity) and to avoid inhibition of respiration by the too high uncoupler concentration. The underlying mechanism for the latter is not clear.
Increasing the concentration in small steps, most accurately titrated by the TIP2k, is recommended (0.5 µM steps or even smaller).
The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentratin, pharmacological treatment (with or without oligomycin), and pathophysiological state (e.g. induction of apoptosis). A single dose of uncoupler usually leads to an artefact in the estmation of maximum flux or electron transfer system capacity.
Discussion
References
- Steinlechner-Maran_1996_AJP: Uncoupler titrations with FCCP and DNP.
- Huetter_2004_BJ: Uncoupler titrations after inhibition of respiration by oligomycin: ROUTINE respiration, LEAK respiration, ETS capacity
- Gnaiger_2008_POS: Uncoupler titrations with the TIP2k (see also O2k-DemoExperiments)
- Pesta 2012 Methods Mol. Biol.: Review of ETS capacity, E, in intact cells, and P/E flux control ratios in permeabilized muscle fibres.
- Gnaiger_2009_IJBCB: Definition of respiratory coupling states in isolated mitochondria and permeabilized cells: L, P, E (see also MitoPathways and Respiratory States)
- O2k-Publications: Coupling; Membrane Potential
