Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Difference between revisions of "Cytochrome c"

From Bioblast
Line 20: Line 20:
::: '''O2k 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 concentration: 10 µM.
::::* Final concentration: 10 µM.


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


The procedures required for [[mitochondrial preparation]]s (homogenization, isolation, permeabilization of the plasma membrane with chemicals, etc) are key steps that determine the quality of the 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 mitochondria isolation or the permeabilization of the cells' plasma membrane 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.
:::: 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 factor|cytochrome ''c'' control factor]]). The following DatLab traces illustrate examples of cytochrome ''c'' addition:


<gallery mode=default perrow=2 widths="800px" 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 factor|cytochrome ''c'' control factor]]=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).


''Performance''
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 factor|cytochrome ''c'' control factor]]=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).
 
It is expected to find stimulation in the respiration rate when cytochrome ''c'' is added, 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 [[permeabilized muscle fibers]] from mice, a significant increase in respiration has been described. If there are 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">
File:cyt_c_traces_O2_flux_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 control factor|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_factor#Cytochrome_c_release|cytochrome ''c'' release]]). BAT tissue homogenate from mouse. EXperiment 2015-07-09-P2-03.
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.
|-
|-
</gallery>
</gallery>


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



Revision as of 13:40, 7 July 2020


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.

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.

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 factor). The following DatLab traces illustrate examples of cytochrome c addition:
See also: Steady state, Figures 1-3.



Questions.jpg


Click to expand or collaps
Bioblast links: DatLab performance and data analysis - >>>>>> - Click on [Expand] or [Collapse] - >>>>>>


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