Difference between revisions of "ADP"
From Bioblast
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{{MitoPedia | {{MitoPedia | ||
|abbr=D | |abbr=D | ||
|description='''Adenosine diphosphate''' is a nucleotid and a substrate of [[ANT]] and [[ATP synthase]]. ADP is the discharged or energy | |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). | ||
|info=[[MiPNet03.02]], [[MiPNet09.12]] | |info=[[MiPNet03.02]], [[MiPNet09.12]] | ||
|type=Substrate PS | |type=Substrate PS | ||
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== Application in [[HRR]] == | == Application in [[HRR]] == | ||
'''D: ADP''' (Adenosine 5'diphosphate potassium salt, C<sub>10</sub>H<sub>15</sub>N<sub>5</sub>O<sub>10</sub>P<sub>2</sub>K) | '''D: ADP''' (Adenosine 5'diphosphate potassium salt, C<sub>10</sub>H<sub>15</sub>N<sub>5</sub>O<sub>10</sub>P<sub>2</sub>K), Ā | ||
MerckĀ 117105-1GM ('''Calbiochem''') FW = 501.3; store at 4Ā°C. Recommended due to lower ATP impurities and used in OROBOROS Lab. | |||
Ā | |||
Alternative source:Ā Sigma A 5285, 1 g, store at -20 Ā°C; FW = 501.3. | |||
<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span> | <span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span> | ||
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'''Preparation of 500 mM ADP stock solution''' (dissolved in H<sub>2</sub>O): | '''Preparation of 500 mM ADP stock solution''' (dissolved in H<sub>2</sub>O): | ||
::1) | ::1) Weigh 501.3 mg of ADP = 1 mmol ADP | ||
::2) | ::2) Add 1.2 ml H<sub>2</sub>OĀ (ADP is not dissolved at this stage). | ||
::3) | ::3) Neutralize with 5 N KOH (approx. 450 Āµl). ADP will dissolve after addition of KOH. | ||
::4) | ::4) Check pH and adjust to 7 if necessary. | ||
::5) | ::5) Adjust final volume to 2 ml and divide into 0.2 ml portions. | ||
::6) | ::6) Store at -80 Ā°C. | ||
'''PreparationĀ of 500 mM ADP stock solution with 3 mM freeĀ Mg<sup>2+</sup>''' (dissolved inĀ H<sub>2</sub>O): | '''PreparationĀ of 500 mM ADP stock solution with 3 mM freeĀ Mg<sup>2+</sup>''' (dissolved inĀ H<sub>2</sub>O): | ||
ToĀ keep freeĀ [Mg<sup>2+</sup>] constant during respirationĀ measurementĀ mix ADP with MgCl<sub>2</sub> (0.6 molĀ MgCl<sub>2</sub>/mol ADP). | ToĀ keep freeĀ [Mg<sup>2+</sup>] constant during respirationĀ measurementĀ in [[MiR06]] or MiR05, mix ADP with MgCl<sub>2</sub> (0.6 molĀ MgCl<sub>2</sub>/mol ADP). | ||
'''MgCl<sub>2</sub>''' (Scharlau MA0036: MgCl<sub>2</sub>.6H<sub>2</sub>O, FW = 203.3) | '''MgCl<sub>2</sub>''' (Scharlau MA0036: MgCl<sub>2</sub>.6H<sub>2</sub>O, FW = 203.3) | ||
::1) | ::1) Weigh 501.3 mg of ADP = 1 mmol ADP. | ||
::2) | ::2) Add 1.2 ml H<sub>2</sub>OĀ (ADP is not dissolved at this stage). | ||
::3) | ::3) Neutralize with 5 N KOH (approx. 450 Āµl). ADP will dissolve after addition of KOH. | ||
::4)Ā | ::4)Ā Add 121,98 mg MgCl<sub>2</sub>. White precipitate will occur, which will dissolve during 1-2 min stirring on magnetic stirrer at RT. | ||
::5) | ::5) Check pH and adjust to 7 if necessary. | ||
::6) | ::6) Adjust final volume to 2 ml and divide into 0.2 ml portions. | ||
::7) | ::7) Store at -80 Ā°C. | ||
Revision as of 15:39, 2 May 2014
- 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 4Ā°C. Recommended due to lower ATP impurities and used in OROBOROS Lab.
Alternative source: Sigma A 5285, 1 g, store at -20 Ā°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 Āµl). 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 Ā°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 Āµl). 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 Ā°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 Āµl using a 25 Āµl syringe (2 ml O2k-chamber).
- Final concentration: 1-5 mM.
