Description
In respiratory assays for cytochrome c oxidase activity (Complex IV, CIV), ascorbate is added as regenerating system for maintaining TMPD in a reduced state. As has to be titrated into the respiration medium prior to the addition of TMPD, otherwise the reaction velocity of autoxidation is permanently elevated.
Abbreviation: As
Reference: MiPNet06.06
MitoPedia topics:
Substrate and metabolite
Application in HRR
- As: Ascorbate sodium salt (Sodium L-ascorbate); Sigma A 4034, 100 g, store at RT; FW = 198.1.
- Caution: Light sensitive (store solution in a dark glass vial).
- Preparation of 800 mM stock solution (dissolved in H2O):
- Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 ml H2O
- To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.ml-1 solution of pH 2).
- Divide into 0.2 ml portions.
- Store frozen at -20 ยฐC.
- O2k manual titrations: MiPNet09.12
- Titration volume: 5 ยตl using a 25 ยตl syringe.
- Final conc. in 2 ml O2k-chamber: 2 mM.