In uncoupler titrations various uncouplers, such as CCCP, FCCP or DNP are applied to uncouple mitochondrial electron transfer from phosphorylation (ATP synthase, ANT and phosphate transporter), particularly with the aim to measure ET-capacity. ET-capacity is maximum oxygen flux measured as noncoupled respiration with optimum uncoupler concentration.
Uncoupler titrations in HRR
- Recommendation: Set "Slope smoothing" to 20 for performing and analyzing experiments with biological sample particularly for uncoupler titrations.
Optimum uncoupler concentration
- Stepwise titrations of an uncoupler is necessary to achive the optimum concentration for obtaining maximum flux as a measure ET-capacity (noncoupled respiration). It is important to avoid inhibition of respiration by too high uncoupler concentrations. The underlying mechanism for the latter is not clear.
- The optimum concentration of an uncoupler has to be determined for every biological system. It varies with incubation medium, sample concentration, 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 estimation of maximum flux or Electron transfer-pathway capacity (for discussion, see Artefacts by single dose uncoupling).
- The optimum uncoupler (CCCP, FCCP, DNP) concentration for the noncoupled state varies over a large concentration range, depending on the medium ('binding' of uncoupler), type and concentration of sample. This is true for various uncouplers, such as CCCP, FCCP and DNP (Steinlechner-Maran 1996 Am J Physiol Cell Physiol). To evaluate the optimum concentration, an uncoupler titration has to be performed initially. For subsequent application series, we recommend a few titrations starting close to optimum concentration (Huetter_2004_Biochem J, Pesta 2012 Methods Mol Biol). Optimum CCCP or FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.
Coupling control protocol
- Uncoupler titrations after inhibition of respiration by oligomycin in coupling control protocols with living cells yield the sequence of ROUTINE respiration, LEAK respiration and ET-capacity, followed by inhibition to ROX (Huetter 2004 Biochem J, Gnaiger 2008 POS). The highest accuracy of uncoupler titrations is achieved by titrations with the TIP2k at high concentrations of the stock solution (Gnaiger 2008 POS. Increasing the concentration in small steps, most accurately titrated by the TIP2k, is recommended (0.5 or 0.25 µM steps or even smaller).
- Bioblast links: Uncoupling - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- » Artefacts by single dose uncoupling
- » ATP synthase
- » CCCP
- » Coupling control protocol
- » DNP
- » Dyscoupled respiration
- » FCCP
- » Gnaiger 2019 MitoFit Preprint Arch
- » Is respiration uncoupled - noncoupled - dyscoupled?
- » Noncoupled respiration: Discussion
- » Uncoupler
- » Uncoupled respiration - see » Noncoupled respiration
- » Uncoupling proteins
- » Uncoupling protein 1
- » Uncoupler titrations - Optimum uncoupler concentration
- Respiratory states and control ratios
- » Biochemical coupling efficiency
- » Coupling control state
- » Electron transfer-pathway state
- » Electron transfer-pathway
- » ET-coupling efficiency
- » Flux control factor
- » Flux control ratio
- » LEAK control ratio
- » LEAK respiration
- » Noncoupled respiration
- » OXPHOS
- » OXPHOS capacity; » State 3
- » OXPHOS control ratio, P/E ratio
- » Respiratory acceptor control ratio
- » ROUTINE control ratio
- » ROUTINE-respiration
- » ROUTINE state
- » State 3u
- » State 4
- » Uncoupler control ratio, UCR
- Respiratory states and control ratios
- Other keyword lists
- Fontana-Ayoub M, Fasching M, Gnaiger E (2016) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(18):1-10. - »Bioblast link«
- Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. - »Bioblast link«
- Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. - »Bioblast link«
- Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58. - »Bioblast link«
- Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. - »Bioblast link«
MitoPedia concepts: MitoFit Quality Control System
MitoPedia methods: Respirometry
MitoPedia topics: Uncoupler