Description
Cytochrome c is a component of the Electron transfer-pathway (Electron transfer pathway) in mitochondria. It is a small heme protein loosely associated with the outer side of the inner mitochondrial membrane. The heme group of cytochrome c transfers electrons from Complex III to Complex IV. The release of cytochrome c into the cytoplasm is associated with apoptosis.
Abbreviation: c
Reference: MiPNet09.12; Gnaiger 2002 Biochem Soc Trans
Application in HRR: storage and stock solution of c
- c: Cytochrome c (from equine heart), Sigma C 7752, 50 mg, store at -20 °C; FW = 12384 Da.
- Preparation of 4 mM cytochrome c solution (dissolved in H2O):
- Weigh 50 mg cytochrome c into a small glass beaker. Difficult to weigh, since the powder is electromagnetic.
- Add 1 mL H2O; c dissolves easily.
- Divide into 0.2 mL portions.
- Store at -20 °C.
- Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
- O2k manual titrations: MiPNet09.12 O2k-Titrations
- Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-Chamber).
- Final concentration: 10 µM.
DatLab oxygen flux: performance and data analysis
- Quality of the results are strongly affected by the performance and data analysis. By adding cytochrome c in respirometric experiments the outer mitochondrial membrane integrity can be evaluated (cytochrome c control factor). The following DatLab traces illustrate examples of cytochrome c addition:
Figure 1. Cytochrome c addition (2c) in OXPHOS state, using pyruvate and malate as NADH-linked substrates. An increase in oxygen flux per volume (red trace, right axis) can be seen upon cytochrome c addition (cytochrome c control factor=0.09). Cardiac isolated mitochondria from mouse. Experiment 2019-02-19 P1-02 (SUIT-008 O2 mt D026, DatLab 7.4).
Figure 2. Example of cytochrome c addition (2c) in OXPHOS state, in the presence of pyruvate and malate as NADH-linked substrates. The (cytochrome c control factor=0.02) indicates that cryopreservation, sample preparation and the use of the detergent digitonin did not affect the integrity of the outer mitochondrial membrane. Cryopreserved HEK-293 cells. Experiment 2017-02-08 P1-02 (SUIT-008 O2 ce-pce D025, DatLab 7.4).
Figure 3. Cytochrome c addition (2c) in OXPHOS state (in the presence of pyruvate, glutamate and malate) triggers an increase of the oxygen flux. This high cytochrome c effect (0.44) may indicate a lost of the outer mitochondrial membrane integrity due to 1) sample preparation or 2) treatment (see cytochrome c release). BAT tissue homogenate from mouse. Experiment 2015-07-09-P2-03.
- See also: Steady state, Figures 1-3.
- For further information see: » Cytochrome c control factor
- See also: Steady state, Figures 1-3.
- Bioblast links: DatLab performance and data analysis - >>>>>> - Click on [Expand] or [Collapse] - >>>>>>
- Performance
- » Smoothing
- » Reoxygenations
- » Steady state
- » Sample addition
- » ROUTINE
- » ADP
- » Cytochrome c
- » Succinate
- » Oligomycin
- » Uncoupler
- » Rotenone
- » Antimycin A
- » Complex IV
- Performance
- Data analysis
- » Smoothing
- » Reoxygenations
- » Steady state
- » Sample addition
- » ROUTINE
- » ADP
- » Cytochrome c
- » Succinate
- » Oligomycin
- » Uncoupler
- » Rotenone
- » Antimycin A
- » Complex IV
- Data analysis
SUITbrowser question: mt outer membrane integrity
- The cytochrome c test is available at several SUIT protocols. The SUITbrowser shows which protocols contain this test, alongside answer other research questions.
MitoPedia topics:
Substrate and metabolite