TMPD

high-resolution terminology - matching measurements at high-resolution

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 required at 0.5 mM final concentration added from a 200 mM stock.

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