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 (Succinate disodium salt, hexahydrate, C6H706Na); 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):
- 1) weigh 1.584 g of ascorbate sodium salt and dissolve in 10 ml H2O
- 2) To prevent autoxidation, adjust pH to ca 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 ยฐC.
Oxygraph-2k manual titrations: MiPNet09.12
- Titration volume: 5 ยตl using a 25 ยตl syringe
- Final conc. in 2 ml O2k-chamber: 2 mM