TMPD

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Bioblasts - Richard Altmann and MiPArt by Odra Noel
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MitoPedia

TMPD

Description

N,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride, TMPD, is applied as an artificial substrate for reducing cytochrome c in the respirometric assay for cytochrome c oxidase (CIV) activity. It is maintained in a reduced state by ascorbate and undergoes autoxidation as a function of oxygen pressure, TMPD, ascorbate and cytochrome c concentration.

Abbreviation: Tm

Reference: MiPNet06.06, MiPNet09.12

Application in HRR

TMPD (C10H16N2*2HCl): Sigma T 3134, 5 g, store at RT; M = 237.2 g·mol-1.
Caution: Sensitive to light and oxidation (store solution in a dark vials). Take care to minimize light, oxygen and temperature exposure during use!
Complex IV assay: For the Complex IV assay TMPD is required at 0.5 mM final concentration added from a 200 mM stock.
During the respirometry experiment it is important to add Ascorbate into the chamber shortly before TMPD to avoid uncontrolled autoxidation. Ascorbate maintains TMPD in a reduced state, and electrons flow from TMPD to cytochrome c. For preparation and titration please visit Ascorbate.
2021-03: The preparation instructions were updated to take the volume of the solute (TMPD) into account (see: Volume_of_the_solute).


Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for 2-mL O2k-chamber:
  1. Weigh 47.4 mg TMPD
  2. Dissolve in 970 µL H2O
  3. Divide into 0.2 mL portions in dark vials
  4. Store frozen at -20 °C
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 5 µL using a 25 µL Hamilton syringe.
  • Final concentration: 0.5 mM.


Preparation of 250 mM stock solution (dissolved in H2O) for 0.5-mL O2k-chamber:
  1. Weigh 59.25 mg TMPD
  2. Dissolve in 962 µL H2O
  3. Divide into 0.2 mL portions in dark vials
  4. Store frozen at -20 °C
» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (0.5-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
  • Final concentration: 0.5 mM.


MitoPedia topics: Substrate and metabolite