Cytochrome c: Difference between revisions

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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 ([[Electron transfer 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'' is applied in [[HRR]] to test the integrity of the [[mitochondrial outer membrane]] ([[cytochrome c control efficiency]]).
|info=[[MiPNet09.12]]; [[Gnaiger_2002_BiochemSocTrans]]
|info=[[MiPNet09.12]]; [[Gnaiger 2002 Biochem Soc Trans]]
|methods_type=Substrate ETS
}}
{{MitoPedia concepts}}
{{MitoPedia methods
|type=Substrate ETS
|type=Substrate ETS
}}
}}
{{MitoPedia O2k and high-resolution respirometry}}
{{MitoPedia topics
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
|mitopedia topic=Substrate and metabolite
|type=Substrate ETS
}}
}}
 
__TOC__
== Application in [[HRR]] ==
== Application in [[HRR]]: storage and stock solution of ''c'' ==
 
{{Chemical_description
''' ''c'': Cytochrome ''c'' ''' (from equine heart), Sigma C 7752, 50 mg, store at -20 °C; FW = 12500
|abbr=c
 
|trivial name=Cytochrome ''c''
 
|complete name=from equine heart
<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span>
|chem formula=small hemeprotein
 
|molar mass=12,384
 
|vendor=Sigma-Aldrich
'''Preparation of 4 mM Cytochrome ''c'' solution''' (dissolved in H<sub>2</sub>O):
|product number=C7752
 
|store at=-20 °C
::1) weigh 50 mg of Cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electromagnetic.
|sensitivity=
::2) add 1 ml H<sub>2</sub>O. Dissolves easily.
|cas=9007-43-6
::3) divide into 0.2 ml portions
|h statements=
::4) store frozen at -20 °C
|h info=
 
}}<!--::: '''''c'': Cytochrome ''c'' ''' (from equine heart), Sigma C 7752, 50 mg, store at -20 °C; MW = 12,384 Da.:::: <span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening. </span>-->::::<span style="color:#2E8B4F">The cytochrome ''c'' recommended (C7752, cytochrome ''c'' from equine heart) is out of stock for the next months (estimated delivery on [https://www.sigmaaldrich.com/AT/en/product/sigma/c7752?context=product November 25, 2021]). The use of other cytochrome ''c'' sources is discussed in the [[Talk:Cytochrome_c|MiPNet discussion forum]].</span>
 
'''Oxygraph-2k Manual Titrations:'''  [[MiPNet09.12]]
 
::* Titration volume: 5 µl using a 25 µl syringe
::* Final conc. in 2 ml O2k-chamber: 10 µM




== Cytochrome ''c'' test for outer mt-membrane integrity ==
:::: '''Preparation of 4 mM cytochrome ''c'' solution''' (dissolved in H<sub>2</sub>O) for '''2-mL O2k-chamber''':


Outer mitochondrial membrane damage can be evaluated from stimulation of respiration by exogenously added cytochrome ''c''.  
::::# Weigh 50 mg cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electrostatically charged.
::::# Add 1 mL H<sub>2</sub>O; ''c'' dissolves easily.
::::# Divide into 0.2 mL portions.
::::# Store at -20 °C.


:::» '''O2k manual titrations:'''  [[MiPNet09.12 O2k-Titrations]]


=== Determination of cytochrome ''c'' loss ===
::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
::::* Final concentration: 10 µM.


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) [[OXPHOS|State ''P'']] or non-coupled (uncoupler stimulated) [[ETS|State ''E'']].


==== Cytochrome ''c'' test ====
:::: '''Preparation of 5 mM cytochrome ''c'' solution''' (dissolved in H<sub>2</sub>O) for '''0.5-mL O2k-chamber''':
::::# Weigh 62.5 mg cytochrome ''c'' into a small glass beaker. Difficult to weigh, since the powder is electrostatically charged.
::::# Add 1 mL H<sub>2</sub>O; ''c'' dissolves easily.
::::# Divide into 0.2 mL portions.
::::# Store at -20 °C.


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?
:::» '''O2k manual titrations:''' [[MiPNet09.12 O2k-Titrations]]


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''.
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
::::* Final concentration: 10 µM.


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.
== DatLab oxygen flux: performance and data analysis ==


Evaluation of the cytochrome ''c'' effect, when respiration is slightly unstable:
:::: Quality of the results are strongly affected by the performance and data analysis. By adding cytochrome ''c'' in respirometric experiments the outer mitochondrial membrane integrity can be evaluated ([[Cytochrome c control efficiency|cytochrome ''c'' control efficiency]]). The following DatLab traces illustrate examples of cytochrome ''c'' addition:
Mark respiration just before cytochrome ''c'' addition and after. Take these to values to calculate the increase of respiration due to cytochrome ''c'' addition.


<gallery mode=default perrow=2 widths="600px" heights="400px">
File:cyt_c_traces_O2_flux_01.png | '''Figure 1'''. Cytochrome ''c'' addition (2c) in OXPHOS state, using pyruvate and malate as NADH-linked substrates. An increase in oxygen flux per volume (red trace, right axis) can be seen upon cytochrome ''c'' addition ([[Cytochrome c control efficiency|cytochrome ''c'' control efficiency]]=0.09). Cardiac isolated mitochondria from mouse. Experiment 2019-02-19 P1-02 ([[SUIT-008_O2_mt_D026|SUIT-008 O2 mt D026]], DatLab 7.4).


==== At which step of the protocol should cytochrome ''c'' be added? ====
File:cyt_c_traces_O2_flux_02.png | '''Figure 2'''. Example of cytochrome ''c'' addition (2c) in  OXPHOS state, in the presence of pyruvate and malate as NADH-linked substrates. The ([[Cytochrome c control efficiency|cytochrome ''c'' control efficiency]]=0.02) indicates that cryopreservation, sample preparation and the use of the detergent [[Digitonin|digitonin]] did not affect the integrity of the outer mitochondrial membrane. Cryopreserved HEK-293 cells. Experiment 2017-02-08 P1-02 ([[SUIT-008_O2_ce-pce_D025|SUIT-008 O2 ce-pce D025]], DatLab 7.4).


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.
File:cyt_c_traces_O2_flux_03.png | '''Figure 3'''. Cytochrome ''c'' addition (2c) in  OXPHOS state (in the presence of pyruvate, glutamate and malate) triggers an increase of the oxygen flux. This high [[Cytochrome c control efficiency|cytochrome ''c'' effect]] (0.44) may indicate a lost of the outer mitochondrial membrane integrity due to 1) sample preparation or 2) treatment (see [[Cytochrome c control efficiency#Cytochrome_c_release|cytochrome ''c'' release]]). BAT tissue homogenate from mouse. Experiment 2015-07-09-P2-03.


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.
File:2019-08-06 P3-02 - A - for cyt c page.png | '''Figure 4'''. Cytochrome ''c'' addition (1c) in  OXPHOS state (in the presence of succinate and rotenone) seems to trigger at first a decrease in oxygen flux, and then an increase up to the same levels as before. It is important to wait for the stabilization of the oxygen flux to set the mark, avoiding an artefactual negative [[Cytochrome c control efficiency|cytochrome ''c'' effect]] (in this case, the cytochrome ''c'' control efficiency was 0.001). Huh 7 cells, permeabilized with digitonin. Experiment 2019-08-06 P3-02.


|-
</gallery>


=== Cytochrome ''c'' release ===
::: See also: [[Steady_state#OXPHOS_state|Steady state]], Figures 1-3.
::::* ''For further information see:'' » [[Cytochrome c control efficiency]]


==== 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''.
{{Keywords: DatLab performance and data analysis}}
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.]]  
== [[SUITbrowser]] question: mt outer membrane integrity ==
#[[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.]]
:::: 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.
*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]

Latest revision as of 09:37, 4 April 2024


high-resolution terminology - matching measurements at high-resolution


Cytochrome c

Description

Cytochrome c is a component of the Electron transfer-pathway (Electron transfer 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 is applied in HRR to test the integrity of the mitochondrial outer membrane (cytochrome c control efficiency).

Abbreviation: c

Reference: MiPNet09.12; Gnaiger 2002 Biochem Soc Trans






MitoPedia topics: Substrate and metabolite 

Application in HRR: storage and stock solution of c

c: Cytochrome c (from equine heart; small hemeprotein), Sigma-Aldrich: C7752, store at -20 °C, CAS: 9007-43-6, M = 12,384 g·mol-1
The cytochrome c recommended (C7752, cytochrome c from equine heart) is out of stock for the next months (estimated delivery on November 25, 2021). The use of other cytochrome c sources is discussed in the MiPNet discussion forum.


Preparation of 4 mM cytochrome c solution (dissolved in H2O) for 2-mL O2k-chamber:
  1. Weigh 50 mg cytochrome c into a small glass beaker. Difficult to weigh, since the powder is electrostatically charged.
  2. Add 1 mL H2O; c dissolves easily.
  3. Divide into 0.2 mL portions.
  4. Store at -20 °C.
» O2k manual titrations: MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 5 µL using a 25 µL Hamilton syringe.
  • Final concentration: 10 µM.


Preparation of 5 mM cytochrome c solution (dissolved in H2O) for 0.5-mL O2k-chamber:
  1. Weigh 62.5 mg cytochrome c into a small glass beaker. Difficult to weigh, since the powder is electrostatically charged.
  2. Add 1 mL H2O; c dissolves easily.
  3. Divide into 0.2 mL portions.
  4. Store at -20 °C.
» O2k manual titrations: MiPNet09.12 O2k-Titrations
  • Titration volume (0.5-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
  • Final concentration: 10 µM.


DatLab oxygen flux: performance and data analysis

Quality of the results are strongly affected by the performance and data analysis. By adding cytochrome c in respirometric experiments the outer mitochondrial membrane integrity can be evaluated (cytochrome c control efficiency). The following DatLab traces illustrate examples of cytochrome c addition:
  • Figure 1. Cytochrome c addition (2c) in OXPHOS state, using pyruvate and malate as NADH-linked substrates. An increase in oxygen flux per volume (red trace, right axis) can be seen upon cytochrome c addition (cytochrome c control efficiency=0.09). Cardiac isolated mitochondria from mouse. Experiment 2019-02-19 P1-02 (SUIT-008 O2 mt D026, DatLab 7.4).

    Figure 1. Cytochrome c addition (2c) in OXPHOS state, using pyruvate and malate as NADH-linked substrates. An increase in oxygen flux per volume (red trace, right axis) can be seen upon cytochrome c addition (cytochrome c control efficiency=0.09). Cardiac isolated mitochondria from mouse. Experiment 2019-02-19 P1-02 (SUIT-008 O2 mt D026, DatLab 7.4).

  • Figure 2. Example of cytochrome c addition (2c) in OXPHOS state, in the presence of pyruvate and malate as NADH-linked substrates. The (cytochrome c control efficiency=0.02) indicates that cryopreservation, sample preparation and the use of the detergent digitonin did not affect the integrity of the outer mitochondrial membrane. Cryopreserved HEK-293 cells. Experiment 2017-02-08 P1-02 (SUIT-008 O2 ce-pce D025, DatLab 7.4).

    Figure 2. Example of cytochrome c addition (2c) in OXPHOS state, in the presence of pyruvate and malate as NADH-linked substrates. The (cytochrome c control efficiency=0.02) indicates that cryopreservation, sample preparation and the use of the detergent digitonin did not affect the integrity of the outer mitochondrial membrane. Cryopreserved HEK-293 cells. Experiment 2017-02-08 P1-02 (SUIT-008 O2 ce-pce D025, DatLab 7.4).

  • Figure 3. Cytochrome c addition (2c) in OXPHOS state (in the presence of pyruvate, glutamate and malate) triggers an increase of the oxygen flux. This high cytochrome c effect (0.44) may indicate a lost of the outer mitochondrial membrane integrity due to 1) sample preparation or 2) treatment (see cytochrome c release). BAT tissue homogenate from mouse. Experiment 2015-07-09-P2-03.

    Figure 3. Cytochrome c addition (2c) in OXPHOS state (in the presence of pyruvate, glutamate and malate) triggers an increase of the oxygen flux. This high cytochrome c effect (0.44) may indicate a lost of the outer mitochondrial membrane integrity due to 1) sample preparation or 2) treatment (see cytochrome c release). BAT tissue homogenate from mouse. Experiment 2015-07-09-P2-03.

  • Figure 4. Cytochrome c addition (1c) in OXPHOS state (in the presence of succinate and rotenone) seems to trigger at first a decrease in oxygen flux, and then an increase up to the same levels as before. It is important to wait for the stabilization of the oxygen flux to set the mark, avoiding an artefactual negative cytochrome c effect (in this case, the cytochrome c control efficiency was 0.001). Huh 7 cells, permeabilized with digitonin. Experiment 2019-08-06 P3-02.

    Figure 4. Cytochrome c addition (1c) in OXPHOS state (in the presence of succinate and rotenone) seems to trigger at first a decrease in oxygen flux, and then an increase up to the same levels as before. It is important to wait for the stabilization of the oxygen flux to set the mark, avoiding an artefactual negative cytochrome c effect (in this case, the cytochrome c control efficiency was 0.001). Huh 7 cells, permeabilized with digitonin. Experiment 2019-08-06 P3-02.

See also: Steady state, Figures 1-3.



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Performance
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» Reoxygenations
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» ROUTINE
» ADP
» Cytochrome c
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» Uncoupler
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» Antimycin A
» Complex IV
Data analysis
» Smoothing
» Reoxygenations
» Steady state
» Sample addition
» ROUTINE
» ADP
» Cytochrome c
» Succinate
» Oligomycin
» Uncoupler
» Rotenone
» Antimycin A
» Complex IV


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.
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