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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.  
|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.
> [[Cytochrome c#Abstract | '''MiPNet article''']]
|info=[[MiPNet09.12]]; [[Gnaiger 2002 Biochem Soc Trans]]
|info=[[MiPNet09.12]]; [[Gnaiger 2002 Biochem Soc Trans]]
|type=Substrate ETS
}}
{{MitoPedia methods|type=Substrate ETS
}}
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
|type=Substrate ETS
}}
}}
__TOC__
__TOC__
== Application in [[HRR]]: storage and stock solution of ''c'' ==
== 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 = 12500.
:::: '''''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 H<sub>2</sub>O):
::: '''Preparation of 4 mM cytochrome ''c'' solution''' (dissolved in H<sub>2</sub>O):


::1. Weigh 50 mg cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electromagnetic.
::::# Weigh 50 mg cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electromagnetic.
::2. Add 1 ml H<sub>2</sub>O; ''c'' dissolves easily.
::::# Add 1 mL H<sub>2</sub>O; ''c'' dissolves easily.
::3. Divide into 0.2 ml portions.
::::# Divide into 0.2 mL portions.
::4. Store at -20 °C.
::::# Store at -20 °C.


'''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
:::: '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.


'''Oxygraph-2k manual titrations:'''  [[MiPNet09.12 O2k-Titrations]]
::: '''O2k manual titrations:'''  [[MiPNet09.12 O2k-Titrations]]


::* Titration volume: 5 µl using a 25 µl syringe (2 ml O2k-chamber)
::::* Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber).
::* Final concwentration: 10 µM
::::* Final concentration: 10 µM.


{{Publication
== Performance and data analysis ==
|title=Gnaiger E (2014) Cytochrome ''c'' control: a test for outer mt-membrane integrity. Mitochondr Physiol Network 2014-04-21.
|info=
|authors=OROBOROS
|year=2014
|journal=Mitochondr Physiol Network
|abstract=The [[cytochrome c control factor | cytochrome ''c'' control factor]], (''J''<sub>CHOc</sub>-''J''<sub>CHO</sub>)/''J''<sub>CHOc</sub>, provides an index for the '''outer mt-membrane integrity'''.
|mipnetlab=AT Innsbruck Gnaiger E
}}
{{Labeling
|instruments=Theory
}}
= Cytochrome ''c'' control: a test for outer mt-membrane integrity =


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


== Determination of cytochrome ''c'' loss ==
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.


Adding cytochrome ''c'' (10 µM)<ref>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. >> [[Kuznetsov_2004_Am_J_Physiol_Heart_Circ_Physiol | '''Open Access''']]</ref> 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 noncoupled (uncoupler stimulated) [[ETS|State ''E'']].
''Analysis''


=== Cytochrome ''c'' test ===
With the cytochrome ''c'' test we will:


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?
#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.


We apply routinely cytochrome ''c'' from Sigma C 7752.<ref>Fontana-Ayoub M, Fasching M, Gnaiger E (2014) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(17): 1-9. >> [[MiPNet03.02 Chemicals-Media | '''Open Access''']]</ref> It would be interesting to compare the consequence of application of different sources of cytochrome ''c''.
::::* ''For further information see:'' » [[Cytochrome c control factor]]


In a detailed discussion on the dependence of respiration in isolated mitochondria and permeabilized cardiac fibers, we showed 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>Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem Soc Trans 30: 242-248. >> [[Gnaiger 2002 Biochem Soc Trans | PMID: 12023860]]</ref>,<ref>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. >> [[Kuznetsov_2004_Am_J_Physiol_Heart_Circ_Physiol | '''Open Access''']]</ref>
== [[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.


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>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. >> [[Renner_2003_Biochim Biophys Acta | PMID: 12972300]]</ref>,<ref>Kuznetsov AV, Gnaiger E (2010) Oxygraph assay of cytochrome c oxidase activity: chemical background correction. Mitochondr Physiol Network 06.06(07): 1-4. >> [[MiPNet06.06 ChemicalBackground | '''Open Access''']]</ref>  Without ascorbate and TMPD, added cytochrome ''c'' is stable.
{{Keywords: DatLab performance and data analysis}}


Evaluation of the cytochrome ''c'' effect, when respiration is slightly unstable:
{{MitoPedia methods|type=Substrate ETS
Mark respiration just before cytochrome ''c'' addition and after. Take these to values to calculate the increase of respiration due to cytochrome ''c'' addition.
}}
 
{{MitoPedia topics
 
|mitopedia topic=Substrate and metabolite
=== 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 ==
<references/>

Revision as of 00:42, 18 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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Performance
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» Reoxygenations
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» Sample addition
» ROUTINE
» ADP
» Cytochrome c
» Succinate
» Oligomycin
» Uncoupler
» Rotenone
» Antimycin A
» Complex IV
Data analysis
» Smoothing
» Reoxygenations
» Steady state
» Sample addition
» ROUTINE
» ADP
» Cytochrome c
» Succinate
» Oligomycin
» Uncoupler
» Rotenone
» Antimycin A
» Complex IV



MitoPedia topics: Substrate and metabolite 

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