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Difference between revisions of "Ascorbate"

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{{MitoPedia
{{MitoPedia
|abbr=As
|abbr=As
|description=In respiratory assays for cytochrome ''c'' oxidase activity ([[Complex IV|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.
|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]]
|info=[[MiPNet06.06]]
}}
}}
{{MitoPedia methods}}
__TOC__
{{MitoPedia topics
 
|mitopedia topic=Substrate and metabolite
== Application in [[HRR]] ==
}}
 
== 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>
<!--
:::: '''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]]


'''As: Ascorbate sodium salt''' (Sodium L-ascorbate); Sigma A 4034, 100 g, store at RT; FW = 198.1.
::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
::::* Final concentration: 2 mM.


<span style="color:#8B008B"> '''Caution:''' Light sensitive (store solution in a dark glass vial).</span>


'''Preparation of 800 mM stock solution''' (dissolved in H<sub>2</sub>O):  
:::: '''Preparation of 1000 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''0.5-mL O2k-chamber''':  


# Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 ml H<sub>2</sub>O
::::# 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 ascorbic acid (a 137.6 mg.ml<sup>-1</sup> solution of pH 2).  
::::# To prevent autoxidation, adjust pH to approx. 6 with 1 M HCl.
# Divide into 0.2 ml portions.  
::::# Adjust final volume to 5 mL and divide into 0.2 mL portions in dark vials.
# Store frozen at -20 °C.  
::::# Store frozen at -20 °C.  


:::»'''O2k manual titrations:''' [[MiPNet09.12]]


'''Oxygraph-2k manual titrations:''' [[MiPNet09.12]]
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
::::* Final concentration: 2 mM.


::* Titration volume: 5 µl using a 25 µl syringe.
{{MitoPedia topics
::* Final conc. in 2 ml O2k-chamber: 2 mM.
|mitopedia topic=Substrate and metabolite
}}

Revision as of 10:15, 22 March 2021


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: 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:
  1. Weigh 792.4 g of ascorbate sodium salt and dissolve in 3 mL H2O
  2. To prevent autoxidation, adjust pH to approx. 6 with 1 M HCl (approx. 85 µL).
  3. Adjust final volume to 5 mL and divide into 0.2 mL portions in dark vials.
  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 990.5 g of ascorbate sodium salt and dissolve in 3 mL H2O
  2. To prevent autoxidation, adjust pH to approx. 6 with 1 M HCl.
  3. Adjust final volume to 5 mL and divide into 0.2 mL portions in dark vials.
  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