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::::# Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube. | ::::# Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube. | ||
::::# Add 180 ยตL H<sub>2</sub>O. | ::::# Add 180 ยตL H<sub>2</sub>O. | ||
:::: '''Preparation of 2.5 M stock solution''' (200 ยตL, dissolved in H<sub>2</sub>O)ย for use in '''0.5-mL O2k-chamber''': | |||
::::# Prepare fresh everyday. | |||
::::# Weigh 55 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube. | |||
::::# Add 175 ยตL H<sub>2</sub>O. | |||
{{Titration instructions | |||
|exp conc=5 mM | |||
|lv titration vol=5 | |||
|lv conc=2 M | |||
|lv syringe vol=25 | |||
|sv titration vol=1 | |||
|sv conc=2.5 M | |||
|sv syringe vol=10 | |||
}} | |||
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:::ยป '''O2k manual titrations'''ย [[MiPNet09.12 O2k-Titrations]] | :::ยป '''O2k manual titrations'''ย [[MiPNet09.12 O2k-Titrations]] | ||
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:::ยป '''O2k manual titrations'''ย [[MiPNet09.12 O2k-Titrations]] | :::ยป '''O2k manual titrations'''ย [[MiPNet09.12 O2k-Titrations]] | ||
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 ยตL using a 10 ยตL Hamilton syringe. | ::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 ยตL using a 10 ยตL Hamilton syringe. | ||
::::* Final concentration: 5 mM. | ::::* Final concentration: 5 mM.--> |
Revision as of 10:46, 21 June 2021
- high-resolution terminology - matching measurements at high-resolution
User:Spitzer Gudrun/Pyruvate test
Description
Pyruvic acid, C3H4O3, is an alpha-keto monocarboxylic acid which occurs under physiological conditions mainly as the anion pyruvate-, P, with pKa = 2.5. Pyruvate is formed in glycolysis from phosphoenolpyruvate. In the cytosol, pyruvate is a substrate of lactate dehydrogenase. Pyruvate enters the mitochondrial matrix via a specific low Km' H+/monocarboxylate cotransporter known as the pyruvate carrier. Similarly, the plasma membrane of many cell types has H+/monocarboxylate cotransporter activity and pyruvate can thus be added as a substrate to living cells. In the mt-matrix the oxidative decarboxylation of pyruvate is catalyzed by pyruvate dehydrogenase and yields acetyl-CoA. Pyruvate competitively reverses the inhibition of cytochrome c oxidase by cyanide. Pyruvate is an antioxidant reacting with hydrogen peroxide.
Abbreviation: P
Reference: Gnaiger 2020 BEC MitoPathways, MiPNet09.12 O2k-Titrations
Application in HRR
- P: Pyruvate (pyruvic acid, sodium salt; C₃H₃O₃Na), Sigma-Aldrich: P2256, store at 4 ยฐC, CAS: 113-24-6, M = 110.0 gยทmol-1
- Hazard statements: H317, H319; may cause an allergic skin reaction, causes serious eye irritation
- P: Pyruvate (pyruvic acid, sodium salt; C₃H₃O₃Na), Sigma-Aldrich: P2256, store at 4 ยฐC, CAS: 113-24-6, M = 110.0 gยทmol-1
- It is possible to weigh the powder beforehand in the Eppendorf-type tubes and store these tubes at 4 ยฐC, to be diluted only on the day of use.
- After addition of H2O the pH of the Pyruvate solution is about 6. This is acceptable without pH-adjustment, because the titrated volumes are small and reaction media are buffered.
- 2021-03: The preparation instructions were updated to take the volume of the solute (P) into account (see: Volume of the solute). The concentrations prepared following the former instructions (see Discussion section) are sufficiently high for SUIT protocol titrations.
- Preparation of 2 M stock solution (200 ยตL, dissolved in H2O) for use in 2-mL O2k-chamber:
- Prepare fresh everyday.
- Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
- Add 180 ยตL H2O.
- Preparation of 2.5 M stock solution (200 ยตL, dissolved in H2O) for use in 0.5-mL O2k-chamber:
- Prepare fresh everyday.
- Weigh 55 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
- Add 175 ยตL H2O.
- ยป O2k manual titrations MiPNet09.12 O2k-Titrations
- Titration volume (2-mL O2k-chamber): 5 ยตL of 2 M stock solution using a 25 ยตL Hamilton syringe.
- Titration volume (0.5-mL O2k-chamber): 1 ยตL of 2.5 M stock solution using a 10 ยตL Hamilton syringe.
- Final concentration: 5 mM.