Description
Adenosine 5βdiphosphate (D), C10H15N5O10P2K, potassium salt; substrate of ANT and ATP synthase.
Abbreviation: D
Reference: MiPNet03.02, MiPNet09.12
MitoPedia topics: Substrate and metabolite
Application in HRR
D: ADP (Adenosine 5'diphosphate, C10H15N5O10P2K, potassium salt); Sigma A 5285, 1 g, store at -20 Β°C): FW = 501.3. Alternative source, that has been recommended for low ATP impurities: Merck 117105-1 (Calbiochem).
Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
Preparation of 500 mM ADP stock solution (dissolved in H2O):
- 1) weigh 501.3 mg of ADP (1 mmol ADP)
- 2) add 1.2 ml H2O (ADP is not dissolved at this stagw.
- 3) neutralize with 5 N KOH (approx. 450 Β΅l) !ADP will dissolve after addition of KOH! (this is Solution A)
- 4) check pH and adjust to 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 ADP stock solution with 3 mM free Mg2+:
To keep free [MgMg2+] constant during respiration measurement mix ADP (or ATP) with MgCl2 (0.6 mol/mol ADP or 0.8 mol/mol ATP).
MgCl2 (Scharlau MA0036: MgCl2.6H2O, FW = 203.3)
- 1) add 121,98 mg MgCl2 to Solution A !White precipitate will occur, which will be dissolved 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 'ADP-Mg' a white precipitate has formed again. If this is the case we recommend to prepare the ADP stock solution and a MgCl2 solution (0.6 mol/mol ADP or 0.8 mol/mol ATP) seperately and perform a titration of both solutions in immediate succession.
Oxygraph-2k Manual Titrations: MiPNet09.12
- Titration volume: 8-20 Β΅l using a 25 Β΅l syringe
- Final conc. in 2 ml O2k-chamber: 2-5 mM