Cytochrome c test for outer membrane integrity
Outer mitochondrial membrane damage can be concluded from stimulation of respiration by exogenously added cytochrome c.
Determination of cytochrome c loss
Adding cytochrome c (10 µM; see Gnaiger_2002_BiochemSocTrans) to stimulate respiratory activity provides an 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 stimulated) State P or non-coupled (uncoupler stimulated) State E.
Cytochrome c test
When using cytochrome c as a quality control for permeabilised muscles from various species, which cytochrome c should we use (a wide range of types of cytochrome c is available from Sigma-Aldrich) and at which concentration?
We apply routinely cytochrome c from Sigma C 7752 (see MiPNet03.02-Selected Media and Chemicals). It would be interesting to compare the consequence of application of different sources of cytochrome c.
A detailed discussion on the dependence of respiration in isolated mitochondria and permeabilized cardiac fibers is found in refs. 1 and 2, for the first time showing that cytochrome c kinetics is different when studying a segment of the ETS (CII-linked: succinate+rotenone) versus the isolated step of cytochrome c oxidase (CIV; ascorbate+TMPD+antimycin A). For CII-linked respiration, an exernal cytochrome c concentration of 10 µM yields kinetic saturation (monophasic hyperbolic), but kinetics is biphasic for CIV and 10 µM is not saturating (ref. 1 and 2).
Importantly, cytochrome c increases the chemical background oxygen flux in the presence of ascorbate and TMPD, dependent on oxygen concentration and cytochrome c concentration, and appropriate chemical background corrections are required (ref. 3). Without ascorbate and TMPD, added cytochrome c is stable.
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.
At which step of the protocol should cytochrome c be added?
If a stimulatory effect of cytochrome c is observed, respiratory capacities measured before cytochrome c addition might be cytochrome c limited and therefore underestimated. This provides an argument to add cytochrome c at an early state of the protocol.
It is important not to add cytochrome c in a LEAK state: There is always an unexplained activation of respiration, unrelated to the injury of the outer mt-membrane. Add cytochrome c only after activation by ADP.
Cytochrome c release
Cytochrome c release induced by sample preparation
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.
Cytochrome c release induced by treatment
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.
References
- Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem. Soc. Trans. 30: 242-248.
- Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. 286: H1633–H1641.
- See also: Renner K, Amberger A, Konwalinka G, Kofler R, Gnaiger E (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim. Biophys. Acta 1642: 115-123.
- Cytochrome c
- Cytochrome c Control
