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 Β°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 Β°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 Β΅L using a 25 Β΅L syringe (2 mL O2k-chamber).
- Final concentration: 10 Β΅M.
Performance and data analysis
The mitochondrial preparation (homogenization, isolation, etc) is one of the key steps that determine the quality of our mitochondrial respiration assessments. During the isolation of the organelles or the permeabilization of the cells/fibers, we must ensure the integrity of the mitochondrial outer membrane. However, some of the techniques used, like the disruption of the tissue for mitochondrial isolation or the permeabilization of the cells plasma membrane with digitonin, could compromise its integrity. For this reason, the addition of cytochrome c is routinely used to test the outer membrane permeability and to evaluate the quality of our mitochondrial preparation. Therefore, the addition of cytochrome c will stimulate respiration if the outer mitochondrial membrane has been altered.
Performance
It is expected to find stimulation in the respiration rate when we add cytochrome c, but the percentage of increase should be determined empirically because it is sample-specific. For example, in the case of HEK 293 cells permeabilized with digitonin, it is well characterized that the cytochrome c has a minimum stimulatory effect in the OXPHOS respiration. However, in other preparations such as muscle fibers from mice, a significant increase in respiration has been described. If there are no data available in the literature for our sample, we need to accumulate enough data and look for consistency.
Figure 1. Example of cytochrome c addition (2c) in OXPHOS state, using pyruvate and malate as substrates, with mitochondria isolated from mouse heart (MiPNet20.15 IsolationRatHeart-mt). An increase in O2 flux (red trace, right axis) can be seen upon cytochrome c addition, which has been consistently seen for this kind of mitochondrial preparation. Image from SUIT-008_O2_mt_D026.DLP, DatLab 7.4.
Figure 2. Example of cytochrome c addition (2c) in OXPHOS state, using pyruvate and malate as substrates, with cryopreserved HEK-293 cells, permeabilized with digitonin in the O2k chamber. Hardly any increase in O2 flux (red trace, right axis) can be seen upon cytochrome c addition, which has been consistently seen for this kind of mitochondrial preparation. Image from SUIT-008_O2_ce-pce_D025.DLP, DatLab 7.4.
- See also: Steady state, Figures 1-3.
Analysis
With the cytochrome c test we will:
- Determine the threshold of the percentage increase to decide if our mitochondrial preparation has good quality and outer membrane integrity.
- Estimate if the mitochondrial capacities before the addition of cytochrome c are underestimated. If there is a stimulatory effect, all the steps before in our protocol have to be considered underestimated.
- For further information see: Β» Cytochrome c control factor
- Bioblast links: DatLab performance and data analysis - >>>>>> - Click on [Expand] or [Collapse] - >>>>>>
- Performance
- Β» Smoothing
- Β» Reoxygenations
- Β» Steady state
- Β» Sample addition
- Β» ROUTINE
- Β» ADP
- Β» Cytochrome c
- Β» Succinate
- Β» Oligomycin
- Β» Uncoupler
- Β» Rotenone
- Β» Antimycin A
- Β» Complex IV
- Performance
- Data analysis
- Β» Smoothing
- Β» Reoxygenations
- Β» Steady state
- Β» Sample addition
- Β» ROUTINE
- Β» ADP
- Β» Cytochrome c
- Β» Succinate
- Β» Oligomycin
- Β» Uncoupler
- Β» Rotenone
- Β» Antimycin A
- Β» 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