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TMPD

From Bioblast


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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

Tm: TMPD (N,N,N′,N′-Tetramethyl-p-phenylenediamine dihydrochloride; C10H16N2*2HCl), Sigma-Aldrich: T3134, store at RT, CAS: 637-01-4, M = 237.2 g·mol-1
Hazard statements: H315, H319; causes skin irritation, causes serious eye irritation
Caution: Sensitive to light and oxidation (store solution in a dark vials). Take care to minimize light, oxygen and temperature exposure during use!
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.
Complex IV assay: For the Complex IV assay TMPD is used at an experimental concentration of 0.5 mM in the O2k-chamber. The abbreviation TMPD is shortened further to the symbol Tm used in DatLab events and SUIT protocol diagrams.
2021-03: The preparation instructions were updated to take into account the volume of the solute (TMPD; see: Volume of the solute).


Preparation of 200 mM stock solution (1 mL, dissolved in H2O) for the 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.
  • Experimental 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.
  • Experimental concentration: 0.5 mM.


MitoPedia topics: Substrate and metabolite