Cytochrome c test for outer membrane integrity
Outer mitochondrial membrane damage can be concluded from stimulation of respiration by exogenously added cytochrome c.
How to determine cytochrome c loss?
Adding cytochrome c to maximum stimulated respiratory activity allows the best estimate of cytochrome c loss. In general, maximum respiratory activity is obtained under conditions of saturating substrate concentrations and system dependent in the coupled (ADP activated) or non-coupled (respiratory activity stimulated by applying an uncoupler) state. If a stimulatory effect of cytochrome c is observed, all respiratory parameters measured before cytochrome c addition might be cytochrome c limited and therefore underestimated.
Sample preparation induced cytochrome c release:
A preparation induced damage of the outer mitochondrial membrane and as a result subsequent loss of cytochrome c can be detected by a stimulation of respiration after the addition of cytochrome c. The preparation induced damage can also affect the respiratory complexes. Therefore, experimental runs showing a preparation induced cytochrome c release should be excluded from the final data set. In perfectly prepared muscle fibers cytochrome c should have no stimulatory effect on maximum respiratory activity, in liver biopsies a small effect is observed, even in carefully prepared samples.
Treatment triggered cytochrome c release, e.g. cell death induction, has to be distinguished from preparation induced damage. If cytochrome c is released as a result of apoptosis induction, this is a biological phenomenon and a relevant result.
Evaluation of the cytochrome c effect, when respiration is slightly unstable: Mark respiration just before cytochrome c addition and after. Take these to values to calculate the increase of respiration due to cytochrome c addition.
See also
Cytochrome c Cytochrome c Control
