Cytochrome c is a component of the Electron transfer-pathway (ET-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.
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.
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 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 the respiration if the outer mitochondrial membrane has been altered.
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 the permeabilized HEK 293 cells, it is well characterized that the cytochrome c has a minimum stimulatory effect in the OXPHOS respiration. However, in other preparations such as the muscle fibers from mice, it has been described a significant increase in the respiration. If there are no data available in the literature for our sample, we need to accumulate enough data and look for consistency.
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: » Cytochrome c control factor
SUITbrowser question: mt outer membrane integrity
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MitoPedia topics: Substrate and metabolite