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.
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.
- Weigh 47.4 mg TMPD
- Dissolve in 1 mL H2O
- Divide into 0.2 mL portions
- Protect from light!
- 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