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!

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.

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.

• 2024-05-03: Ascorbate should be added to the TMPD stock solution to maintain TMPD in a reduced state and protect it from oxidation. This was the procedure until 2014, when addition of ascorbate to the TMPD stock was discontinued for unknown reasons, as noticed in May 2024, when instructions were changed again to prepare and use the TMPD stock solution containing ascorbate.

• 2021-03-9: The preparation instructions were updated to take into account the volume of the solute (TMPD; see: Volume of the solute).

2024-05-03 to present

Preparation of 200 mM stock solution (1 mL, dissolved in H₂O, with ascrobate) for the 2-mL O2k-chamber:

1. Weigh 47.4 mg TMPD
2. Dissolve in 970 mL H₂O
3. Add 0.8 M ascorbate to a final concentration of 10 mM
4. Divide into 0.2 mL portions
5. 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. Add 0.8 M ascorbate to a final concentration of 10 mM
4. Divide into 0.2 mL portions in dark vials
5. 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.

2021-03-09 to 2024-05-03

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

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

2014-03-20 to 2021-03-09

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 1 mL H₂O
3. Divide into 0.2 mL portions in dark vials
4. Store frozen at -20 °C

up to 2014-03-20

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 1 mL H₂O
3. Add 0.8 M ascorbate to a final concentration of 10 mM
4. Divide into 0.2 mL portions in dark vials
5. Store frozen at -20 °C

MitoPedia topics: Substrate and metabolite