Ascorbate

high-resolution terminology - matching measurements at high-resolution

Ascorbate

Description

In respiratory assays for cytochrome c oxidase activity (Complex IV, CIV), ascorbate is added as regenerating system to maintain TMPD in a reduced state. It has to be titrated into the respiration medium prior to the addition of TMPD, otherwise the autoxidation reaction velocity is permanently elevated.

Abbreviation: As

Reference: MiPNet06.06

Application in HRR

As: Ascorbate sodium salt (Sodium L-ascorbate); Sigma A 7631, 100 g, store at RT; M = 198.1 g·mol^-1.

Caution: Light sensitive (store solution in a dark glass vial).

Preparation of 800 mM stock solution (dissolved in H₂O) for 2-mL O2k-chamber:

1. Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 mL H₂O
2. To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL^-1 solution of pH 2).
3. Divide into 0.2 mL portions.
4. Store frozen at -20 °C.

»O2k manual titrations: MiPNet09.12

• Titration volume (2-mL O2k-chamber): 5 µL using a 25 µL Hamilton syringe.
• Final concentration: 2 mM.

Preparation of 1000 mM stock solution (dissolved in H₂O) for 0.5-mL O2k-chamber:

1. Weigh 1.980 g of ascorbate sodium salt and dissolve in 10 mL H₂O
2. To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL^-1 solution of pH 2).
3. Divide into 0.2 mL portions.
4. Store frozen at -20 °C.

»O2k manual titrations: MiPNet09.12

• Titration volume (0.5-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
• Final concentration: 2 mM.

MitoPedia topics: Substrate and metabolite