Difference between revisions of "Rotenone"
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== Application in [[HRR]] == | == Application in [[HRR]] == | ||
:::'''Rot: Rotenone''' (C<sub>23</sub>H<sub>22</sub>O<sub>6</sub>); Sigma R 8875, 1 g, store commercial stock at RT; | :::'''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:''' 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''': | :::: '''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%). | ::::# 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). | ::::# Divide into 100 µL aliquots (use dark glass vials). | ||
::::# Store frozen at -20 °C. | ::::# Store frozen at -20 °C. | ||
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:::: '''Preparation of 0.25 mM stock solution''' (dissolved in 99.9% ethanol) for '''0.5-mL O2k-chamber''': | :::: '''Preparation of 0.25 mM stock solution''' (dissolved in 99.9 % ethanol) for '''0.5-mL O2k-chamber''': | ||
::::# Take 50 µL of the 1 mM stock solution. | ::::# Take 50 µL of the 1 mM stock solution. | ||
::::# Add 150 µL of ethanol absolute (99.9%). | ::::# Add 150 µL of ethanol absolute (99.9 %). | ||
:::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | :::» '''O2k manual titrations''' [[MiPNet09.12 O2k-Titrations]] | ||
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::: 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: | ||
:::: '''Preparation of 5.5 mM stock solution''' (dissolved in 99.9% ethanol) | :::: '''Preparation of 5.5 mM stock solution''' (dissolved in 99.9 % ethanol) | ||
::::# Weigh 4.4 mg of rotenone and dissolve in 2 ml of ethanol absolute (99.9%). | ::::# 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). | ::::# Divide into 200 µl aliquots (use dark glass vials). | ||
::::# Store frozen at -20 °C. | ::::# Store frozen at -20 °C. | ||
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<!--::::* Titration volume: 15 µL using a 25 µL syringe (2 mL O2k-Chamber).--> | <!--::::* 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. | ::::* Titration volume ('''2-mL O2k-chamber'''): 15 µL using a 25 µL Hamilton syringe. | ||
::::* | ::::* Experimental concentration: 41 µM. | ||
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::::* 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. | |||
{{MitoPedia topics | {{MitoPedia topics | ||
|mitopedia topic=Inhibitor | |mitopedia topic=Inhibitor | ||
}} | }} |
Revision as of 12:41, 12 February 2021
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:
- 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:
- Take 50 µL of the 1 mM stock solution.
- 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)
- 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 (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