Difference between revisions of "Rotenone"

Latest revision as of 10:13, 5 March 2024

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 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

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.

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |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 centers in CI to ubiquinone via binding to the ubiquinone binding site of CI. See also [[Complex II-linked substrate state |Complex II-linked substrate state, S<sub>(Rot)</sub>]].
+|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 2014 MitoPathways]], [[MiPNet09.12]], [[MiPNet03.02]]
+|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]] ==
+{{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>
-::::'''Rot: Rotenone''' (C<sub>23</sub>H<sub>22</sub>O<sub>6</sub>); Sigma R 8875, 1 g, store commercial stock at RT; FW = 394.42 (≥ 95%).
-:::: <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 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.
-::: '''Preparation of 1 mM stock solution''' (dissolved in 99.9% ethanol)
+:::» '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
-::::# Weigh 0.39 mg of rotenone and dissolve in 1 ml of ethanol absolute (99.9%).
+::::* Titration volume ('''2-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
-::::# Divide into 100 µl aliquots (use dark glass vials).
+::::* Final concentration: 0.5 µM.
-::::# Store frozen at -20 °C.
+:::: '''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 µl using a 10 µl syringe (2 ml O2k-chamber).
+::::* 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%).
+:::: '''Preparation of 5.5 mM stock solution''' (dissolved in ethanol abs.)
-::::# Divide into 200 µl aliquots (use dark glass vials).
+::::# 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]]
-::: '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
+<!--::::* 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.
-::::* Titration volume: 15 µL using a 25 µL syringe (2 mL O2k-Chamber).
-::::* Final 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. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2724837/ >>PMC2724837]
-{{MitoPedia concepts}}
+== History ==
-{{MitoPedia methods}}
-{{MitoPedia O2k and high-resolution respirometry}}
+::::* 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 topic=Inhibitor
-}}