Ascorbate: Difference between revisions

Revision as of 06:25, 8 January 2016

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Ascorbate

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 °C.

Oxygraph-2k manual titrations: MiPNet09.12

Titration volume: 5 µl using a 25 µl syringe.
Final conc. in 2 ml O2k-chamber: 2 mM.

@@ Line 16: / Line 16: @@
 '''Preparation of 800 mM stock solution''' (dissolved in H<sub>2</sub>O):
-::1) weigh 1.584 g of ascorbate sodium salt and dissolve in 10 ml H<sub>2</sub>O
+# Weigh 1.584 g of ascorbate sodium salt and dissolve in 10 ml H<sub>2</sub>O
-::2) 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 ascorbic acid (a 137.6 mg.ml<sup>-1</sup> solution of pH 2).
-::3) Divide into 0.2 ml portions.
+# Divide into 0.2 ml portions.
-::4) Store frozen at -20 °C.
+# Store frozen at -20 °C.
 '''Oxygraph-2k manual titrations:''' [[MiPNet09.12]]
-::* Titration volume: 5 µl using a 25 µl syringe
+::* Titration volume: 5 µl using a 25 µl syringe.
-::* Final conc. in 2 ml O2k-chamber: 2 mM
+::* Final conc. in 2 ml O2k-chamber: 2 mM.