Rotenone

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 about 1 mg of rotenone.
2. Calculate the amount of ethanol abs. needed: 1 mL of ethanol abs. per 0.39 mg of rotenone.
3. Rotenone does not dissolve easily in ethanol. Warming to 35°C and an ultrasonic bath will improve solubility.
4. Divide into 100 µL aliquots (use dark glass vials).
5. 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