Difference between revisions of "DTPA"

Revision as of 14:44, 22 August 2022

high-resolution terminology - matching measurements at high-resolution

DTPA

Description

DTPA (Diethylenetriamine-N,N,N',N,N-pentaacetic acid, pentetic acid,(Carboxymethyl)imino]bis(ethylenenitrilo)-tetra-acetic acid) is a polyaminopolycarboxylic acid (like EDTA) chelator of metal cations. DTPA wraps around a metal ion by forming up to eight bounds, because each COO- group and and N-center serves a center for chelation. With transition metals the number of bounds is less than eight. The compound is not cell membrane permeable. In general, it chelates multivalent ions stronger than EDTA.

Abbreviation: DTPA

Application in HRR

Fluorometry:

In Amplex UltraRed assay systems with HRP, there is a spontaneous increase in the UltoroxRed fluorescence signal over the experimental time without any biological sample. This high background flux can be related to iron contamination. We have done a set of experiments, where we added DTPA as an iron-chelator into the O2k-chamber during AmR measurements. As a matter of fact, DTPA significantly reduced the high chemical background flux in MiR05 without affecting respiration.
Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation of the compound is time-dependent; it takes 10-15 min.
Experiments may also be run without DTPA.

DTPA (C₂H₂O₂); Dr.Ehrenstorfer GmbH (E 13095000) or Sigma Aldrich (D-1133); CAS 67-43-6; The commercial powder can be stored at room temperature. The stock solution has to be stored at -20 °C; M: 393.35 g·mol^-1

Solubility: It is soluble in water (4,8 g/l at 25 °C), but not readily soluble in ethanol or apolar solvents.
Stability constant: see: [1]:

Metal	DTPA	EDTA
Ca(II)	10.74	10.96
Mg(II)	9.3	8.69
Fe(II)	16.5	14.3
Fe(III)	28.6	25.1
Mn(II)	15.6	14.04
Zn(II)	18.75	16.5

Preparation of the 5 mM stock solution of DTPA:

Weigh 1.967 mg of DTPA in a 2 mL glass vial.
Dissolve in 500 µL H₂O and add 6 µL of 5 M KOH to set the pH.
Heat up the solution up to 33 °C.
Add 2 µL 5 M KOH to adjust the pH ~ 8-9.
Add 500 µL H₂O to get 5 mM stock solution.
Divide into 50 µL portions in Eppendorf tubes.
Store at -20 °C.

»O2k manual titrations: MiPNet09.12 O2k-Titrations

Final concentration in O2k-chamber: 15 µM
Stock solution: 5 mM
Titration volume: 6 µL

MitoPedia methods: Fluorometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=DTPA
-|description='''DTPA''' (Diethylentriamin-N,N,N',N,N-penta acetic acid, "Triplex V") is a chelator of transition metals, not cell membrane permeable.
+|description='''DTPA''' (Diethylenetriamine-N,N,N',N,N-pentaacetic acid, pentetic acid,(Carboxymethyl)imino]bis(ethylenenitrilo)-tetra-acetic acid) is a polyaminopolycarboxylic acid (like EDTA) chelator of metal cations. DTPA wraps around a metal ion by forming up to eight bounds, because each COO- group and and N-center serves a center for chelation.  With transition metals the number of bounds is less than eight. The compound is not cell membrane permeable. In general, it chelates multivalent ions stronger than EDTA.
 }}
+[[File:DTPA structure.gif|right|200px|DTPA structure]]
+__TOC__
-== Description ==
+== Application in [[HRR]] ==
+:::'''Fluorometry:'''
+::::* In [[Amplex UltraRed]] assay systems with [[HRP]], there is a spontaneous increase in the UltoroxRed fluorescence signal over the experimental time without any biological sample. This high background flux can be related to iron contamination. We have done a set of experiments, where we added DTPA as an iron-chelator into the O2k-chamber during AmR measurements. As a matter of fact, DTPA significantly reduced the high chemical background flux in MiR05 without affecting respiration.
+::::* Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation of the compound is time-dependent; it takes 10-15 min.
+::::* Experiments may also be run without DTPA.
+:::'''DTPA''' (C<sub>2</sub>H<sub>2</sub>O<sub>2</sub>); Dr.Ehrenstorfer GmbH (E 13095000) or Sigma Aldrich (D-1133); CAS 67-43-6; The commercial powder can be stored at room temperature. The stock solution has to be stored at -20 °C; M: 393.35 g·mol<sup>-1</sup>
-::::* CAS: 67-43-6
+::::* '''Solubility:''' It is soluble in water (4,8 g/l at 25 °C), but not readily soluble in ethanol or apolar solvents.
-::::* Source: EHERE13095000	VWR 0.25 g
+::::* '''Stability constant:''' see: [http://www.dojindo.com/Images/Product%20Photo/Chelate_Table_of_Stability_Constants.pdf]:
-::::* M: 393,35 g/mol
+:::::{| class="wikitable"
-::::* Stability constants not included in NIST version of Martell 1989, constants from [http://www.dojindo.com/Images/Product%20Photo/Chelate_Table_of_Stability_Constants.pdf]: Ca2+: 10.74 (EDTA 10.96), Mg2+: 9.3 (EDTA: 8.69) Fe II 16.5 (EDTA 14.3), Fe III 28.6 (EDTA 25.1) Mn II 15.6 (EDTA 14.04), ZN 18.75 (EDTA 16.5), general chelates multivalent ions stronger than EDTA; pKa1 = 2.14, pKa2 = 2.38, pKa3 = 4.26 pKa4 = 8.60, pKa5 = 10.53
+|-
-::::* Solubility in H2O: ca 4,8 g·l−1 bei 25 °C = 12 mM
+!Metal !!  DTPA !! EDTA
+|-
+|Ca(II)|| 10.74 ||10.96
+|-
+|Mg(II)|| 9.3 ||8.69
+|-
+|Fe(II)|| 16.5 ||14.3
+|-
+|Fe(III) || 28.6 ||25.1
+|-
+|Mn(II) || 15.6 ||14.04
+|-
+|Zn(II) || 18.75 || 16.5
+|-
+|}
+::: '''Preparation of the 5 mM stock solution of DTPA:'''
-== DTPA in O2k-FluoRespirometry ==
+::::# Weigh 1.967 mg of DTPA in a 2 mL glass vial.
+::::# Dissolve in 500 µL H<sub>2</sub>O and add 6 µL of 5 M KOH to set the pH.
+::::# Heat up the solution up to 33 °C.
+::::# Add 2 µL 5 M KOH to adjust the pH ~ 8-9.
+::::# Add 500 µL H<sub>2</sub>O to get 5 mM stock solution.
+::::# Divide into 50 µL portions in Eppendorf tubes.
+::::# Store at -20 °C.
+:::»'''O2k manual titrations:''' [[MiPNet09.12 O2k-Titrations]]
 ::::* Final concentration in O2k-chamber: 15 µM
-::::* Stock solution:
+::::* Stock solution: 5 mM
+::::* Titration volume: 6 µL
+{{MitoPedia methods
+|mitopedia method=Fluorometry
+}}
+{{MitoPedia O2k and high-resolution respirometry}}
+{{MitoPedia topics}}