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Difference between revisions of "Rotenone"

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{{MitoPedia
{{MitoPedia
|abbr=Rot
|abbr=Rot
|description=(2R,6aS,12aS)-1,2,6,6a,12,12a-hexahydro-2-isopropenyl-8,9 dimethoxychromeno[3,4-b]furo(2,3-h)chromen-6-on; C<sub>23</sub>H<sub>22</sub>O<sub>6</sub>. Inhibitor of [[complex I]] via inhibition of NADH oxidation.It inhibits the transfer of electrons from iron-sulfur centers in CI to ubiquinone via binding to the ubiquinone binding site of CI. See also [[S(Rot)|S<sub>(Rot)</sub>]].
|description='''Rotenone''' is an inhibitor of [[Complex I]] (CI) and thus inhibits NADH oxidation. It inhibits the transfer of electrons from iron-sulfur clusters in CI to ubiquinone via binding to the ubiquinone binding site of CI. See also [[Complex II-linked substrate state |Complex II-linked substrate state, S<sub>(Rot)</sub>]].
|info=[[MiPNet11.09]], [[MiPNet09.12]], [[MiPNet12.15]], [[MiPNet03.02]]
|info=[[Gnaiger 2020 BEC MitoPathways]], [[MiPNet09.12]], [[MiPNet03.02]]
|type=Inhibitor ETS
}}
{{Labeling
|discipline=Mitochondrial Physiology
|enzymes=Complex I
|kinetics=Inhibitor; Uncoupler
|instruments=Chemicals; Media
|type=Inhibitor ETS
}}
}}
__TOC__
== Application in [[HRR]] ==
== Application in [[HRR]] ==


'''Rot: Rotenone''' (Rotenone ≥ 95%, C<sub>23</sub>H<sub>22</sub>O<sub>6</sub>); Sigma R 8875, 1 g, store at R.T.: FW = 394.42)
:::'''Rot: Rotenone''' (C<sub>23</sub>H<sub>22</sub>O<sub>6</sub>); Sigma R 8875, 1 g, store commercial stock at RT; M = 394.42 g·mol<sup>-1</sup> (≥ 95%).
 
:::: <span style="color:#8B008B"> '''Caution:''' Very toxic and light sensitive (store solution in a dark glass vial).</span>
 
 
:::: '''Preparation of 1 mM stock solution''' (dissolved in 99.9 % ethanol) for '''2-mL O2k-chamber''':
 
::::# Weigh 0.39 mg of rotenone and dissolve in 1 ml of ethanol absolute (99.9 %).
::::# Divide into 100 µL aliquots (use dark glass vials).
::::# Store frozen at -20 °C.
 
:::» '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
 
::::* Titration volume ('''2-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
::::* Final concentration: 0.5 µM.
 
 
:::: '''Preparation of 0.25 mM stock solution''' (dissolved in 99.9 % ethanol) for '''0.5-mL O2k-chamber''':


<span style="color:#8B008B"> '''Caution:''' Very toxic and light sensitive (store solution in a dark glass vial).</span>
::::# Take 50 µL of the 1 mM stock solution.
::::# Add 150 µL of ethanol absolute (99.9 %).


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


'''Preparation of 0.1 mM stock solution''' <ref>Note: Up to 2006, rotentone was added at a higher final concentration 0.5 µM), hence a 0.5 mM stock solution was prepared. Since 0.05 µM may be fully inhibiting, we suggest to use a stock solution at lower concentration (0.1 mM), to reduce the problem or rotenone retention.</ref> (dissolved in 99.9% Ethanol):
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
::::* Final concentration: 0.5 µM.


::1) weigh 0.39 mg of Rotenone and dissolve in 10 ml of absolute ethanol
::2) divide into small portions (use dark glass vials)
::3) store frozen at -20 °C


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


::* Titration volume: 1 µl using a 10 µl syringe
:::'''Microalgae applications'''
::* Final conc. in 2 ml O2k-chamber: 0.5 µM
::: For the specific case of microalgal cells it is recommended to prepare higher concentration:


:::: '''Preparation of 5.5 mM stock solution''' (dissolved in 99.9 % ethanol)


<references/>
::::# Weigh 4.4 mg of rotenone and dissolve in 2 ml of ethanol absolute (99.9 %).
::::# Divide into 200 µl aliquots (use dark glass vials).
::::# Store frozen at -20 °C.
 
:::» '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
 
<!--::::* Titration volume: 15 µL using a 25 µL syringe (2 mL O2k-Chamber).-->
::::* Titration volume ('''2-mL O2k-chamber'''): 15 µL using a 25 µL Hamilton syringe.
::::* Experimental concentration: 41 µM.
 
 
== Inhibition of CI by rotenone ==
::::* Fato R, Bergamini C, Bortolus M, Maniero AL, Leoni S, Ohnishi T, Lenaz G (2009) Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species. Biochim Biophys Acta 1787:384–92. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2724837/ >>PMC2724837]
 
== History ==
 
::::* Up to 2006, rotenone was added at a high final concentration (0.5 µM), hence a 0.5 mM stock solution was prepared. Since 0.05 µM may be fully inhibiting, we suggested to use a stock solution at lower concentration (0.1 mM), to reduce the problem of rotenone retention. The 2006 edition listed a 0.2 mM stock solution, but the amount added to 10 ml ethanol was given for a 0.15 mM stock solution. Our recent rotenone titrations with permeabilized muscle fibers, however, confirmed that the originally proposed higher rotenone concentration is actually required for full inhibition.
 
{{MitoPedia topics
|mitopedia topic=Inhibitor
}}

Revision as of 11:41, 12 February 2021


high-resolution terminology - matching measurements at high-resolution


Rotenone

Description

Rotenone is an inhibitor of Complex I (CI) and thus inhibits NADH oxidation. It inhibits the transfer of electrons from iron-sulfur clusters in CI to ubiquinone via binding to the ubiquinone binding site of CI. See also Complex II-linked substrate state, S(Rot).

Abbreviation: Rot

Reference: Gnaiger 2020 BEC MitoPathways, MiPNet09.12, MiPNet03.02

Application in HRR

Rot: Rotenone (C23H22O6); Sigma R 8875, 1 g, store commercial stock at RT; M = 394.42 g·mol-1 (≥ 95%).
Caution: Very toxic and light sensitive (store solution in a dark glass vial).


Preparation of 1 mM stock solution (dissolved in 99.9 % ethanol) for 2-mL O2k-chamber:
  1. Weigh 0.39 mg of rotenone and dissolve in 1 ml of ethanol absolute (99.9 %).
  2. Divide into 100 µL aliquots (use dark glass vials).
  3. Store frozen at -20 °C.
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
  • Final concentration: 0.5 µM.


Preparation of 0.25 mM stock solution (dissolved in 99.9 % ethanol) for 0.5-mL O2k-chamber:
  1. Take 50 µL of the 1 mM stock solution.
  2. Add 150 µL of ethanol absolute (99.9 %).
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (0.5-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
  • Final concentration: 0.5 µM.


Microalgae applications
For the specific case of microalgal cells it is recommended to prepare higher concentration:
Preparation of 5.5 mM stock solution (dissolved in 99.9 % ethanol)
  1. Weigh 4.4 mg of rotenone and dissolve in 2 ml of ethanol absolute (99.9 %).
  2. Divide into 200 µl aliquots (use dark glass vials).
  3. Store frozen at -20 °C.
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 15 µL using a 25 µL Hamilton syringe.
  • Experimental concentration: 41 µM.


Inhibition of CI by rotenone

  • Fato R, Bergamini C, Bortolus M, Maniero AL, Leoni S, Ohnishi T, Lenaz G (2009) Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species. Biochim Biophys Acta 1787:384–92. >>PMC2724837

History

  • Up to 2006, rotenone was added at a high final concentration (0.5 µM), hence a 0.5 mM stock solution was prepared. Since 0.05 µM may be fully inhibiting, we suggested to use a stock solution at lower concentration (0.1 mM), to reduce the problem of rotenone retention. The 2006 edition listed a 0.2 mM stock solution, but the amount added to 10 ml ethanol was given for a 0.15 mM stock solution. Our recent rotenone titrations with permeabilized muscle fibers, however, confirmed that the originally proposed higher rotenone concentration is actually required for full inhibition.


MitoPedia topics: Inhibitor