User:Spitzer Gudrun/Pyruvate test

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 pK_a = 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 K_m' 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

General

P: Pyruvate (pyruvic acid, sodium salt; C₃H₃O₃Na), Sigma-Aldrich: P2256, store at 4 °C, CAS: 113-24-6, M = 110.0 g·mol^-1

Hazard statements: H317, H319; may cause an allergic skin reaction, causes serious eye irritation

It is possible to weigh the powder beforehand in the Eppendorf-type tubes and store these tubes at 4 °C, 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 into account the volume of the solute (P) (see: Volume of the solute). The concentrations prepared following the former instructions (see Discussion section) are sufficiently high for SUIT protocol titrations.

Preparation of stock solution

2-mL O2k-chamber: 200 µL in H₂O

1. Prepare fresh everyday.
2. Weigh 44 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
3. Add 180 µL H₂O.

0.5-mL O2k-chamber: 200 µL in H₂O

1. Prepare fresh everyday.
2. Weigh 55 mg of pyruvic acid directly into a 0.5 mL Eppendorf tube.
3. Add 175 µL H₂O.

Manual titrations

» O2k manual titrations MiPNet09.12 O2k-Titrations

• Titration volume (2-mL O2k-chamber): 5 µL of 2 M stock solution using a 25 µL Hamilton syringe.
• Titration volume (0.5-mL O2k-chamber): 1 µL of 2.5 M stock solution using a 10 µL Hamilton syringe.
• Final concentration: 5 mM.