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Revision as of 11:12, 22 March 2021 by Spitzer Gudrun (talk | contribs) (corrected instructions: added volume adjustment, halved the amound of stock solution prepared, corrected that we adjust pH with HCl not ascorbic acid. Added info of air and light sensitivity)
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: Solutions of ascorbic acid are rapidly oxidized in air and in alkaline media; the compound should be protected from air and light. See 'ascorbid acid' on PubChem
- Preparation of 800 mM stock solution (dissolved in H2O) for 2-mL O2k-chamber:
- Weigh 792.4 g of ascorbate sodium salt and dissolve in 3 mL H2O
- To prevent autoxidation, adjust pH to approx. 6 with 1 M HCl (approx. 85 µL).
- Adjust final volume to 5 mL and divide into 0.2 mL portions in dark vials.
- 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:
- Weigh 990.5 g of ascorbate sodium salt and dissolve in 3 mL H2O
- To prevent autoxidation, adjust pH to approx. 6 with 1 M HCl.
- Adjust final volume to 5 mL and divide into 0.2 mL portions in dark vials.
- 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