Difference between revisions of "TMPD"

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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:

Weigh 47.4 mg TMPD
Dissolve in 970 µL H₂O
Divide into 0.2 mL portions in dark vials
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:

Weigh 59.25 mg TMPD
Dissolve in 962 µL H₂O
Divide into 0.2 mL portions in dark vials
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

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=Tm
-|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 [[Complex IV|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.
+|description='''''N,N,N',N'''-Tetramethyl-''p''-phenylenediamine dihydrochloride, TMPD''', is applied as an artificial substrate for reducing [[cytochrome c|cytochrome ''c'']] in the respirometric assay for [[Complex IV|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.
 |info=[[MiPNet06.06]], [[MiPNet09.12]]
-|type=Substrate ETS
-}}
-{{MitoPedia methods
-|mitopedia method=Respirometry
-|type=Substrate ETS
-}}
-{{MitoPedia topics
-|mitopedia topic=Substrate and metabolite
-|type=Substrate ETS
 }}
 __TOC__
 == Application in [[HRR]]  ==
-=== Complex IV assay ===
+{{Chemical_description
-* For the  [[Complex_IV#HRR_protocol:_Complex_IV_assay|Complex IV assay]] TMPD is  required at 0.5 mM final concentration added from a 200 mM stock.
+|abbr=Tm
+|trivial name=TMPD
+|complete name=''N,N,N′,N′''-Tetramethyl-''p''-phenylenediamine dihydrochloride
+|chem formula=C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl
+|molar mass=237.2
+|vendor=Sigma-Aldrich
+|product number=T3134
+|store at=RT
+|sensitivity=
+|cas=637-01-4
+|h statements=H315, H319
+|h info=causes skin irritation, causes serious eye irritation
+}}<!--::: '''TMPD''' (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl): Sigma T 3134, 5 g, store at RT; M =237.2  g·mol<sup>-1</sup>.-->
+:::: <span  style="color:#8B008B"> '''Caution:''' Sensitive to light and oxidation (store  solution in a dark vials). Take care to minimize light, oxygen and temperature exposure during use!</span>
+:::: 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#HRR_protocol:_Complex_IV_assay|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 H<sub>2</sub>O) for the '''2-mL O2k-chamber''':
+::::# Weigh 47.4 mg TMPD
+::::# Dissolve in 970 µL H<sub>2</sub>O
+::::# Divide into 0.2 mL portions in dark vials
+::::# Store frozen at -20 °C
+:::» '''O2k manual titrations '''  [[MiPNet09.12 O2k-Titrations]]
-=== Preparation of 200 mM stock ===
+::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
-TMPD    (C<sub>10</sub>H<sub>16</sub>N<sub>2</sub>*2HCl):   Sigma T 3134, 5 g, store at RT; FW = 237.2.
+::::* Experimental concentration: 0.5 mM.
-::1) Weigh 47.4 mg TMPD
-::2) Dissolve in 1 ml H<sub>2</sub>O
-::3) Divide into 0.2 ml portions
-::4) Protect from light!
-::5) Store frozen at -20 °C
-<span  style="color:#8B008B"> '''Caution:''' Light sensitive (store  solution in a dark glass vial). Take care to minimize light and  temperature exposure during use!</span>
-It is  important to add  [[Ascorbate]] before TMPD  to avoid  uncontrollable  autoxidation.  Ascorbate maintains TMPD in a  reduced  state. Electrons  flow from TMPD to cytochrome c.
+::::''' Preparation of 250 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''0.5-mL O2k-chamber''':
-For preparation and titration please visit [[Ascorbate]]
+::::# Weigh 59.25 mg TMPD
+::::# Dissolve in 962 µL H<sub>2</sub>O
+::::# Divide into 0.2 mL portions in dark vials
+::::# Store frozen at -20 °C
+:::» '''O2k manual titrations '''  [[MiPNet09.12 O2k-Titrations]]
-'''Oxygraph-2k Manual Titrations:'''  [[MiPNet09.12]]
+::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
+::::* Experimental concentration: 0.5 mM.
-::* Titration volume: 5 µl using a 25 µl syringe
-::* Final conc. in 2 ml O2k-chamber: 0.5 mM
+{{MitoPedia topics
+|mitopedia topic=Substrate and metabolite
+}}