Rotenone

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Bioblasts - Richard Altmann and MiPArt by Odra Noel
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MitoPedia

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