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Carbonyl cyanide m-chlorophenyl hydrazone

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(Redirected from CCCP)


high-resolution terminology - matching measurements at high-resolution


Carbonyl cyanide m-chlorophenyl hydrazone

Description

Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U; C9H5ClN4; FW = 204.62) is a protonophore (H+ ionophore) and is used as a potent chemical uncoupler of oxidative phosphorylation. Like all uncouplers, CCCP concentrations must be titrated carefully to evaluated the optimum concentration for maximum stimulation of mitochondrial respiration, particularly to avoid inhibition of respiration at higher CCCP concentrations.

Abbreviation: CCCP

Reference: Lou 2007 Biochem J, Skulachev 1998 Biochim Biophys Acta


Application in HRR

CCCP: m-Cl-CCP (Carbonyl cyanide 3-chlorophenylhydrazone; C9H5ClN4O), Sigma-Aldrich: C2759, store at -20 ยฐC, CAS: 555-60-2, M = 204.62 gยทmol-1
Hazard statements: H301, H311, H331, H315, H319, H335; toxic if swallowed, toxic in contact with skin, toxic if inhaled, causes skin irritation, causes serious eye irritation, may cause respiratory irritation
Solubility: CCCP also can be prepared in DMSO as well as EtOH. Soluble in DMSO (5 mg/mL), ethanol (1 mg/mL), and methanol (10 mg/mL). Insoluble in water.
Because of better handling at room temperature, we use EtOH in OROBOROS Lab.
Disadvantage of DMSO: freezing point of +18.55 ยฐC (65.4 ยฐF). When using DMSO as solvent, make sure to bring the stock solution to room temperature before filling the 10 ยตL syringe, which is important to avoid blocking the needle.
Ethanol stock solutions may be stored in 5 mL glass vials (instead of freezing 0.2 mL portions in small vials). Use subsamples during experiments to avoid contamination of the storage solution.
Many labs routinely protect CCCP from light. However, there is no hint on vendor's pages that this is necessary (see Discussion).
NOTE: 0.1 mM stock for mt-preparations with high uncoupler sensitivity; 1 mM stock for mt-preparations with low uncoupler sensitivity, living cells in various culture media (e.g., RPMI, DMEM, EGC) and for TIP2k.


Preparation of 1 mM stock solution (dissolved in Ethanol) for 2-mL O2k-chamber:
  1. Weigh 1.02 mg of CCCP into a glass vial.
  2. Dissolve in 5 mL ethanol.
  3. Divide into 0.2 mL portions in glass vials and store at -20 ยฐC.
ยป O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 1 ยตL (or 0.5 ยตL) steps using a 10 ยตL Hamilton syringe.
  • Final concentration: 0.5 ยตM (or 0.25 ยตM) steps


Preparation of 0.25 mM stock solution (dissolved in Ethanol) for 0.5-mL O2k-chamber:
  1. Take 100 ยตL of 1 mM stock solution.
  2. Dilute with 300 ยตL ethanol.
ยป O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (0.5-mL O2k-chamber): 1 ยตL (or 0.5 ยตL) steps using a 10 ยตL Hamilton syringe.
  • Final concentration: 0.5 ยตM (or 0.25 ยตM) steps


MiPNet News CCCP 2019-05-02

CCCP solution is less yellow coloured than FCCP. This may be important when working with fluorometric applications in parallel with high-resolution respirometry. - Cardoso LHD

MiPNet News CCCP 2014-01-15

CCCP is soluble in ethanol up to 10 mM. Working with CCCP below room temperature is therefore possible. - Heidler J

MiPNet News CCCP 2014-01-10

The temperature of the CCCP stock solution dissolved in DMSO should be increased to room temperature before filling the titration syringe. When the CCCP stock solution is kept on ice, the needle of the Hamilton titration syringe can be easily blocked, causing problems during titrations which may not be detected immediately. When performing experiments below room temperature, the needle of the Hamilton syringe is regularly blocked during injections into the O2k chamber. To avoid such problems, FCCP should be used instead of CCCP at low experimental temperature, or in laboratories at low room temperature that may prevail during expeditions. - Laner V, Gnaiger E

MiPNet News CCCP 2013-07-11

  • We replace FCCP by CCCP in the OROBOROS Mitochondrial Research Laboratory. Up to now, we used and recommended FCCP as an uncoupler in high-resolution respirometry. Recently we compared the uncoupling effect of CCCP and FCCP in our on-going experiments with homogenate preparations of mouse brain and heart. In parallel experiments identical results were obtained on ET capacity and mt-membrane potential. Compared to FCCP, a much higher DNP concentration is required for maximum stimulation of oxygen flux (Steinlechner-Maran 1996 Am J Physiol Cell Physiol). In contrast, the optimum CCCP concentration for maximum oxygen flux was only slightly higher than the optimum FCCP concentration. CCCP is significantly less expensive than FCCP. - Eigentler A, Gnaiger E
  • Bioblast alert 2013(02)






MitoPedia topics: Uncoupler 


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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