Difference between revisions of "Ascorbate"
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{{MitoPedia | {{MitoPedia | ||
|abbr=As | |abbr=As | ||
|description=Ascorbate sodium salt, | |description=In respiratory assays for cytochrome ''c'' oxidase activity ([[Complex IV|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. | ||
|info=[[MiPNet06.06]] | |||
| | }} | ||
__TOC__ | |||
== Application in [[HRR]] == | |||
:::: '''As: Ascorbate sodium salt''' (Sodium L-ascorbate); Sigma A 7631, 100 g, store at RT; M = 198.1 g·mol<sup>-1</sup>. | |||
:::: <span style="color:#8B008B"> '''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 [https://pubchem.ncbi.nlm.nih.gov/compound/54670067 PubChem]</span> | |||
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:::: '''Preparation of 800 mM stock solution''' (dissolved in H<sub>2</sub>O): | |||
::::# Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 mL H<sub>2</sub>O | |||
::::# To prevent autoxidation, adjust pH to approx. 6 with ascorbic acid (a 137.6 mg.mL<sup>-1</sup> solution of pH 2). | |||
::::# Divide into 0.2 mL portions. | |||
::::# Store frozen at -20 °C. | |||
:::» '''O2k manual titrations:''' [[MiPNet09.12]] | |||
::::* Titration volume: 5 µL using a 25 µL syringe. | |||
::::* Final conc. in 2 mL O2k-chamber: 2 mM. | |||
--> | |||
:::: '''Preparation of 800 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''2-mL O2k-chamber''': | |||
::::# Weigh 792.4 g of ascorbate sodium salt and dissolve in 3 mL H<sub>2</sub>O | |||
::::# 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 H<sub>2</sub>O) for '''0.5-mL O2k-chamber''': | |||
::::# Weigh 990.5 g of ascorbate sodium salt and dissolve in 3 mL H<sub>2</sub>O | |||
::::# 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 | |||
|mitopedia topic=Substrate and metabolite | |||
}} | }} | ||
Revision as of 11:15, 22 March 2021
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