Rotenone: Difference between revisions
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:::: '''Preparation of 5.5 mM stock solution''' (dissolved in ethanol abs.) | :::: '''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 | ::::# Weigh 4.4 mg of rotenone and dissolve in 2 mL of ethanol abs. | ||
::::# Divide into 200 ยตL aliquots (use dark glass vials). ย | ::::# Divide into 200 ยตL aliquots (use dark glass vials). ย | ||
::::# Store frozen at -20 ยฐC. | ::::# Store frozen at -20 ยฐC. |
Latest 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 gยทmol-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 gยทmol-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 35ยฐC and an ultrasonic bath will improve solubility.
- Divide into 100 ยตL aliquots (use dark glass vials).
- Store frozen at -20 ยฐC.
- ยป O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 1 ยตL using a 10 ยตL Hamilton syringe.
- Final concentration: 0.5 ยตM.
- Preparation of 0.25 mM stock solution (dissolved in ethanol abs.) for 0.5-mL O2k-chamber:
- Take 50 ยตL of the 1 mM stock solution.
- Add 150 ยตL of ethanol abs.
- ยป O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (0.5-mL O2k-chamber): 1 ยตL using a 10 ยตL Hamilton syringe.
- Final concentration: 0.5 ยตM.
- 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 ยตL aliquots (use dark glass vials).
- Store frozen at -20 ยฐC.
- ยป O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 15 ยตL using a 25 ยตL Hamilton syringe.
- Experimental concentration: 41 ยตM.
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 1787:384โ92. >>PMC2724837
History
- Up to 2006, rotenone was added at a high final concentration (0.5 ยตM), hence a 0.5 mM stock solution was prepared. Since 0.05 ยตM 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.