Cytochrome c

high-resolution terminology - matching measurements at high-resolution

Cytochrome c

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):

1. Weigh 50 mg cytochrome c into a small glass beaker. Difficult to weigh, since the powder is electromagnetic.
2. Add 1 mL H₂O; c dissolves easily.
3. Divide into 0.2 mL portions.
4. 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

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

Data analysis

» Smoothing

» Reoxygenations

» Steady state

» Sample addition

» ROUTINE

» ADP

» Cytochrome c

» Succinate

» Oligomycin

» Uncoupler

» Rotenone

» Antimycin A

» Complex IV

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