Description
Adenosine triphosphate is a nucleotid and functions as the major carrier of chemical energy in the cells. As it transfers its energy to other molecules, it looses its terminal phosphate group and becomes adenosine diphosphate (ADP).
Abbreviation: T
Reference: MiPNet03.02; MiPNet09.12
MitoPedia topics: Substrate and metabolite
Application in HRR
T: ATP (Adenosine 5'-triphosphate disodium salt hydrate, C10H14N5O13P3Na2* (H2O)x); Sigma A 2383, 5 g, store at -20 Β°C; FW = 614.1 containing 3.5 mol H2O/mol (FWanhydrous basis = 551.1). Alternative source: Merck 1191-1GM (Calbiochem, Adenosine 5β²-Triphosphate Disodium Salt; FW = 551.2; store at -20 Β°C).
Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
Preparation of 500 mM ATP stock solution
- 1) Weigh 614.1 mg of ATP (FW = 614.1 containing 3.5 mol H2O/mol).
- 2) Add 1.2 ml H2O
- 3) Neutralize with 5 N KOH (approx. 400 Β΅l) - ATP dissolves after addition of KOH (this is Solution A).
- 4) Check pH and adjust to pH 7 if necessary.
- 5) Adjust final volume to 2 ml and divide into 0.2 ml portions.
- 6) Store frozen at -80 Β°C.
Preparation of 500 mM ATP stock solution with 3 mM free Mg2+
To keep free [MgMg2+] constant during respiration measurement in MiR05 or MiR06, mix ATP with MgCl2 (0.8 mol MgCl2 /mol ATP).
MgCl2 (Scharlau MA0036: MgCl2.6H2O, FW = 203.3)
- 1) Add 162.64 mg MgCl2 to Solution A. A white precipitate forms, which dissolves after 1-2 min of stirring on magnetic stirrer at room temperature.
- 2) Follow steps 4-6 of the above instructions.
- Comment: In some cases it was observed, that after defreezing of the 'ATP-Mg' a white precipitate forms again. If this is the case we recommend to prepare the 500 mM ATP stock solution and a MgCl2 solution (0.8 mol MgCl2 /mol ATP) seperately and perform a titration of both solutions in series.
Oxygraph-2k manual titrations
- Titration volume: 4-20 Β΅l using a 25 Β΅l syringe.
- Final conc. in 2 ml O2k-chamber: 1-5 mM.