ADP

high-resolution terminology - matching measurements at high-resolution

ADP

Description

Adenosine diphosphate is a nucleotid and a substrate of ANT and ATP synthase. ADP is the discharged or low-energy counterpart of ATP. ADP can accept chemical energy by regaining a phosphate group to become ATP, in substrate-level phosphorylation (in anaerobic catabolism), at the expense of solar energy (in photosynthetic cells) or chemical energy (respiration in heterotrophic cells).

Abbreviation: D

Reference: MiPNet03.02, MiPNet09.12

MitoPedia methods: Respirometry 

MitoPedia topics: Substrate and metabolite 

Application in HRR

D: ADP (Adenosine 5'diphosphate potassium salt, C₁₀H₁₅N₅O₁₀P₂K), Merck 117105-1GM (Calbiochem) FW = 501.3; store at 4°C. Recommended due to lower ATP impurities and used in OROBOROS Lab.

Alternative source: Sigma A 5285, 1 g, store at -20 °C; FW = 501.3.

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 stage).

3) Neutralize with 5 N KOH (approx. 450 µl). ADP will dissolve after addition of KOH.

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 at -80 °C.

Preparation of 500 mM ADP stock solution with 3 mM free Mg²⁺ (dissolved in H₂O):

To keep free [Mg²⁺] constant during respiration measurement in MiR06 or MiR05, mix ADP with MgCl₂ (0.6 mol MgCl₂/mol ADP).

MgCl₂ (Scharlau MA0036: MgCl₂.6H₂O, FW = 203.3)

1) Weigh 501.3 mg of ADP = 1 mmol ADP.

2) Add 1.2 ml H₂O (ADP is not dissolved at this stage).

3) Neutralize with 5 N KOH (approx. 450 µl). ADP will dissolve after addition of KOH.

4) Add 121,98 mg MgCl₂. White precipitate will occur, which will dissolve during 1-2 min stirring on magnetic stirrer at RT.

5) Check pH and adjust to 7 if necessary.

6) Adjust final volume to 2 ml and divide into 0.2 ml portions.

7) Store at -80 °C.

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 MgCl₂/mol ADP) seperately and perform a titration of both solutions in immediate succession.

Oxygraph-2k manual titrations MiPNet09.12 O2k-Titrations

• Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber).
• Final concentration: 1-5 mM.