Rotenone: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr=Rot | |abbr=Rot | ||
|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 [[ | |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 [[Succinate pathway]]. | ||
|info=[[Gnaiger | |info=[[Gnaiger 2020 BEC MitoPathways]], [[MiPNet03.02]] | ||
}} | }} | ||
{{MitoPedia concepts}} | |||
{{MitoPedia methods}} | |||
{{MitoPedia O2k and high-resolution respirometry}} | |||
{{MitoPedia topics | |||
|mitopedia topic=Inhibitor | |||
}} | |||
__TOC__ | |||
== Application in [[HRR]] == | == Application in [[HRR]] == | ||
{{Chemical_description | |||
|abbr=Rot | |||
|trivial name=Rotenone | |||
|complete name= | |||
|chem formula=C<sub>23</sub>H<sub>22</sub>O<sub>6</sub> | |||
|molar mass=394.42 | |||
|vendor=Sigma-Aldrich | |||
|product number=R8875 | |||
|store at=RT | |||
|sensitivity=light sensitive | |||
|cas=83-79-4 | |||
|h statements=H300, H330, H315, H319, H335, H410 | |||
|h info=fatal if swallowed, fatal if inhaled, causes skin irritation, causes serious eye irritation, may cause respiratory irritation, very toxic to aquatic life with long-lasting effects}}<!--:::'''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>--> | |||
:::: <span style="color:#8B008B"> '''Caution:''' In solution and exposed to light and air, rotenone decomposes losing toxicity within days, see [https://pubchem.ncbi.nlm.nih.gov/compound/6758#section=Stability-Shelf-Life PubChem] for details.</span> | |||
:::: | :::: '''Preparation of 1 mM stock solution''' (dissolved in ethanol abs.) for '''2-mL O2k-chamber''': | ||
::::# Weigh about 1 mg of rotenone. | |||
::::# Calculate the amount of ethanol abs. needed: 1 mL of ethanol abs. per 0.39 mg of rotenone. | |||
::::# Rotenone does not dissolve easily in ethanol. Warming to 35°C and an ultrasonic bath will improve solubility. | |||
::::# 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 ethanol abs.) for '''0.5-mL O2k-chamber''': | |||
::::# Take 50 µL of the 1 mM stock solution. | |||
::::# Add 150 µL of ethanol abs. | |||
::: '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | :::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | ||
::::* Titration volume: 1 | ::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe. | ||
::::* Final concentration: 0.5 µM. | ::::* Final concentration: 0.5 µM. | ||
:::'''Microalgae applications''' | :::'''Microalgae applications''' | ||
::: For the specific case of microalgal cells it is recommended to prepare higher concentration: | ::: For the specific case of microalgal cells it is recommended to prepare higher concentration: | ||
::::# Weigh 4.4 mg of rotenone and dissolve in 2 | :::: '''Preparation of 5.5 mM stock solution''' (dissolved in ethanol abs.) | ||
::::# Divide into 200 | |||
::::# Weigh 4.4 mg of rotenone and dissolve in 2 mL of ethanol abs. | |||
::::# Divide into 200 µL aliquots (use dark glass vials). | |||
::::# Store frozen at -20 °C. | ::::# Store frozen at -20 °C. | ||
:::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | |||
::: '''O2k | <!--::::* 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 == | == 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] | ::::* 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. | |||
Revision as of 10:13, 5 March 2024
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 Succinate pathway.
Abbreviation: Rot
Reference: Gnaiger 2020 BEC MitoPathways, MiPNet03.02
MitoPedia topics: Inhibitor
Application in HRR
- Rot: Rotenone ( C23H22O6), Sigma-Aldrich: R8875, store at RT (light sensitive), CAS: 83-79-4, M = 394.42 g·mol-1
- Hazard statements: H300, H330, H315, H319, H335, H410; fatal if swallowed, fatal if inhaled, causes skin irritation, causes serious eye irritation, may cause respiratory irritation, very toxic to aquatic life with long-lasting effects
- Rot: Rotenone ( C23H22O6), Sigma-Aldrich: R8875, store at RT (light sensitive), CAS: 83-79-4, M = 394.42 g·mol-1
- Caution: In solution and exposed to light and air, rotenone decomposes losing toxicity within days, see PubChem for details.
- Preparation of 1 mM stock solution (dissolved in ethanol abs.) for 2-mL O2k-chamber:
- Weigh about 1 mg of rotenone.
- Calculate the amount of ethanol abs. needed: 1 mL of ethanol abs. per 0.39 mg of rotenone.
- Rotenone does not dissolve easily in ethanol. Warming to 35°C and an ultrasonic bath will improve solubility.
- 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 ethanol abs.) for 0.5-mL O2k-chamber:
- Take 50 µL of the 1 mM stock solution.
- Add 150 µL of ethanol abs.
- » 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 ethanol abs.)
- Weigh 4.4 mg of rotenone and dissolve in 2 mL of ethanol abs.
- Divide into 200 µL aliquots (use dark glass vials).
- 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.