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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-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.
|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 Biochem Soc Trans]]
|info=[[MiPNet09.12]]; [[Gnaiger 2002 Biochem Soc Trans]]
}}
}}
__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 = 12384 Da.
{{Chemical_description
|abbr=c
|trivial name=Cytochrome ''c''
|complete name=from equine heart
|chem formula=small hemeprotein
|molar mass=12,384
|vendor=Sigma-Aldrich
|product number=C7752
|store at=-20 °C
|sensitivity=
|cas=9007-43-6
|h statements=
|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>




::: '''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) for '''2-mL O2k-chamber''':


::::# 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 electrostatically charged.
::::# Add 1 mL H<sub>2</sub>O; ''c'' dissolves easily.
::::# Add 1 mL H<sub>2</sub>O; ''c'' dissolves easily.
::::# Divide into 0.2 mL portions.
::::# Divide into 0.2 mL portions.
::::# 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.
:::» '''O2k manual titrations:''' [[MiPNet09.12 O2k-Titrations]]


::: '''O2k manual titrations:''' [[MiPNet09.12 O2k-Titrations]]
::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
 
::::* Titration volume: 5 µL using a 25 µL syringe (2 mL O2k-chamber).
::::* Final concentration: 10 µM.
::::* 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 respiration if the outer mitochondrial membrane has been altered.  
:::: '''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.


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


''Performance''
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
::::* Final concentration: 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 HEK 293 cells permeabilized with digitonin, it is well characterized that the cytochrome ''c'' has a minimum stimulatory effect in the OXPHOS respiration. However, in other preparations such as muscle fibers from mice, a significant increase in respiration has been described. If there is no data available in the literature for our sample, we need to accumulate enough data and look for consistency.


<gallery mode=default perrow=2 widths="800px" heights="400px">
== DatLab oxygen flux: performance and data analysis ==
File:D026 O2 traces.png | '''Figure 1'''. Example of cytochrome ''c'' addition (2c) in  OXPHOS state, using pyruvate and malate as substrates, with mitochondria isolated from mouse heart ([[MiPNet20.15 IsolationRatHeart-mt]]). An increase in O<sub>2</sub> flux (red trace, right axis) can be seen upon cytochrome ''c'' addition, which has been consistently seen for this kind of mitochondrial preparation. Image from SUIT-008_O2_mt_D026.DLP, DatLab 7.4.


File:D025 O2 traces.png | '''Figure 2'''. Example of cytochrome ''c'' addition (2c) in  OXPHOS state, using pyruvate and malate as substrates, with cryopreserved HEK-293 cells, permeabilized with digitonin in the O2k chamber. Hardly any increase in O<sub>2</sub> flux (red trace, right axis) can be seen upon cytochrome ''c'' addition, which has been consistently seen for this kind of mitochondrial preparation. Image from SUIT-008_O2_ce-pce_D025.DLP, DatLab 7.4.
:::: 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:
|-
</gallery>


::: See also: [[Steady_state#OXPHOS_state|Steady state]], Figures 1-3.
<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).


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).


''Analysis''
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.


With the cytochrome ''c'' test we will:
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.


# 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.
</gallery>


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




{{Keywords: DatLab performance and data analysis}}
{{Keywords: DatLab performance and data analysis}}





Latest revision as of 15:43, 14 September 2021


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

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:
See also: Steady state, Figures 1-3.



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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.




MitoPedia topics: Substrate and metabolite