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Bioblasts - Richard Altmann and MiPArt by Odra Noel
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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

MitoPedia topics: Substrate and metabolite 

Application in HRR

Complex IV assay

  • For the Complex IV assay TMPD is required at 0.5 mM final concentration added from a 200 mM stock.

Preparation of 200 mM stock

TMPD (C10H16N2*2HCl): Sigma T 3134, 5 g, store at RT; FW = 237.2.
  1. Weigh 47.4 mg TMPD
  2. Dissolve in 1 mL H2O
  3. Divide into 0.2 mL portions
  4. Protect from light!
  5. Store frozen at -20 °C
Caution: Light sensitive (store solution in a dark glass vial). Take care to minimize light and temperature exposure during use!

Oxygraph-2k Manual Titrations: MiPNet09.12
  • Titration volume: 5 µL using a 25 µL syringe.
  • Final conc. in 2 mL O2k-chamber: 0.5 mM.
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. Electrons flow from TMPD to cytochrome c.
For preparation and titration please visit Ascorbate