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Uncoupler titrations

From Bioblast


high-resolution terminology - matching measurements at high-resolution


Uncoupler titrations

Description

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 carrier), particularly with the aim to measure ET capacity. ET capacity is maximum oxygen flux measured as noncoupled respiration with optimum uncoupler concentration.



MitoPedia methods: Respirometry 


MitoPedia O2k and high-resolution respirometry: DatLab, Oroboros QM, O2k-Open Support 


MitoPedia topics: Uncoupler 

Uncoupler titrations in HRR

DatLab settings

  • 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 ยตM.
See Steinlechner-Maran et al (1996) for a comparison of uncoupler titrations with FCCP and DNP from the ROUTINE state to the ET state of cell respiration.

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 ยตM steps or even smaller).


References

Bioblast linkReferenceYear
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 & Sons, Inc, Hoboken, NJ:327-52.2008
Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. https://doi.org/10.1016/j.biocel.2009.03.0132009
Hรผtter E, Renner K, Pfister G, Stรถckl P, Jansen-Dรผrr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. https://doi.org/10.1042/BJ200400952004
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. https://doi.org/10.1007/978-1-61779-382-0_32012
Selected media and chemicals for respirometry with mitochondrial preparations.
O2k-Protocols
2016-08-30
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:C2053-61.1996
Bioblast linkReferenceYear
Gnaiger E et al โ€• MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v12020


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Specific
ยป Artefacts by single dose uncoupling
ยป ATP synthase
ยป CCCP
ยป Coupling-control protocol
ยป DNP
ยป Dyscoupled respiration
ยป FCCP
ยป Is respiration uncoupled - noncoupled - dyscoupled?
ยป Noncoupled respiration: Discussion
ยป Uncoupler
ยป Uncoupled respiration - see ยป Noncoupled respiration
ยป Uncoupling proteins
ยป Uncoupling protein 1
ยป Uncoupler titrations - Optimum uncoupler concentration
Respiratory states and control ratios
ยป Biochemical coupling efficiency
ยป Coupling-control state
ยป Electron-transfer-pathway state
ยป Electron-transfer pathway
E.jpg ET capacity
ยป E-L coupling efficiency
ยป Flux control efficiency
ยป Flux control ratio
ยป LEAK-control ratio
ยป LEAK respiration
ยป Noncoupled respiration
ยป OXPHOS
ยป OXPHOS capacity; ยป State 3
ยป OXPHOS-control ratio, P/E ratio
ยป Respiratory acceptor control ratio
ยป ROUTINE-control ratio
ยป ROUTINE respiration
ยป ROUTINE state
ยป State 3u
ยป State 4
ยป Uncoupling-control ratio UCR
General (alphabetical order)
ยป Adenine nucleotide translocase
ยป Adenylates
ยป Electron transfer pathway
ยป Mitochondrial preparations
ยป mt-membrane potential
ยป Oxygen flux
ยป Phosphorylation system
ยป Proton leak
ยป Proton slip
ยป TIP2k
Other keyword lists
ยป Template:Keywords: Force and membrane potential