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Difference between revisions of "Cytochrome c"

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{{MitoPedia
{{MitoPedia
|abbr=''c''
|abbr=c
|description='''Cytochrome ''c''''' is a component of the electron transfer system ([[ETS]]) 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. [http://en.wikipedia.org/wiki/Cytochrome_c].
|description='''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.
|info=[[MiPNet09.12]]; [[Gnaiger 2002 Biochem Soc Trans]]
|info=[[MiPNet09.12]]; [[Gnaiger 2002 Biochem Soc Trans]]
|type=Substrate ETS
}}
}}
{{MitoPedia methods|type=Substrate ETS
__TOC__
}}
== Application in [[HRR]]: storage and stock solution of ''c'' ==
{{MitoPedia topics
:::: '''''c'': Cytochrome ''c'' ''' (from equine heart), Sigma C 7752, 50 mg, store at -20 °C; FW = 12384 Da.
|mitopedia topic=Substrate and metabolite
|type=Substrate ETS
}}
== Application in [[HRR]] ==


''' ''c'': Cytochrome ''c'' ''' (from equine heart), Sigma C 7752, 50 mg, store at -20 °C; FW = 12500


::: '''Preparation of 4 mM cytochrome ''c'' solution''' (dissolved in H<sub>2</sub>O):


<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span>
::::# Weigh 50 mg cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electromagnetic.
::::# Add 1 mL H<sub>2</sub>O; ''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.


'''Preparation of 4 mM Cytochrome ''c'' solution''' (dissolved in H<sub>2</sub>O):
::: '''O2k manual titrations:''' [[MiPNet09.12 O2k-Titrations]]


::1) weigh 50 mg of Cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electromagnetic.
::::* Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber).
::2) add 1 ml H<sub>2</sub>O. Dissolves easily.
::::* Final concentration: 10 µM.
::3) divide into 0.2 ml portions
::4) store frozen at -20 °C


== Performance and data analysis ==


'''Oxygraph-2k Manual Titrations:''' [[MiPNet09.12]]
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.  


::* Titration volume: 5 µl using a 25 µl syringe
''Performance''
::* Final conc. in 2 ml O2k-chamber: 10 µM


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.


== Cytochrome ''c'' test for outer mt-membrane integrity ==
''Analysis''


Outer mitochondrial membrane damage can be evaluated from stimulation of respiration by exogenously added cytochrome ''c''.
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.


=== Determination of cytochrome ''c'' loss ===
::::* ''For further information see:'' » [[Cytochrome c control factor]]


Adding cytochrome ''c'' (10 µM; see [[Gnaiger 2002 Biochem Soc Trans]]) 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) [[OXPHOS|State ''P'']] or non-coupled (uncoupler stimulated) [[ETS|State ''E'']].  
== [[SUITbrowser]] question: mt outer membrane integrity ==
:::: The cytochrome ''c'' test is available at several [[SUIT]] protocols. The [https://suitbrowser.oroboros.at/ SUITbrowser] shows which protocols contain this test, alongside answer other research questions.


==== Cytochrome ''c'' test ====
{{Keywords: DatLab performance and data analysis}}


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?
{{MitoPedia methods|type=Substrate ETS
 
}}
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''.
{{MitoPedia topics
 
|mitopedia topic=Substrate and metabolite
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_2002_BiochemSocTrans|Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem. Soc. Trans. 30: 242-248.]]
#[[Kuznetsov_2004_AJP|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_2003_BBA|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]]
*[http://www.oroboros.at/index.php?cytochromeccontrol Cytochrome ''c'' control]

Revision as of 23:42, 17 February 2020


high-resolution terminology - matching measurements at high-resolution


Cytochrome c

Description

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.

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.

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.

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 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.

Analysis

With the cytochrome c test we will:

  1. Determine the threshold of the percentage increase to decide if our mitochondrial preparation has good quality and outer membrane integrity.
  2. 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.

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.


Questions.jpg


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MitoPedia topics: Substrate and metabolite