Difference between revisions of "ADP"

Revision as of 18:02, 6 November 2016

high-resolution terminology - matching measurements at high-resolution

ADP

Description

Adenosine diphosphate is a nucleotid. In OXPHOS core metabolism, ADP is a substrate of ANT and ATP synthase in the phosphorylation system. 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 chemiosmotic energy (respiration in heterotrophic cells). ADP is added to mitochondrial preparations at kinetically saturating concentrations to induce the active state for evaluation of OXPHOS capacity.

Abbreviation: D

Reference: MiPNet03.02, MiPNet09.12

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, we use 'ADP-Mg' 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):

Weigh 501.3 mg of ADP = 1 mmol ADP
Add 1.2 ml H₂O (ADP is not dissolved at this stage).
Neutralize with 5 M KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
Check pH and adjust to 7 if necessary.
Adjust final volume to 2 ml and divide into 0.2 ml portions.
Store at -80 °C.

Preparation of 500 mM ADP stock solution with 300 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)

Weigh 501.3 mg of ADP = 1 mmol ADP.
Add 1.2 ml H₂O (ADP is not dissolved at this stage).
Neutralize with 5 M KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
Add 121,98 mg MgCl₂. White precipitate will occur, which will dissolve during 1-2 min stirring on magnetic stirrer at RT.
Check pH and adjust to 7 if necessary.
Adjust final volume to 2 ml and divide into 0.2 ml portions.
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.

O2k 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.

@@ Line 3: / Line 3: @@
 |description='''Adenosine diphosphate''' is a nucleotid. In [[OXPHOS]] core metabolism, ADP is a substrate of [[ANT]] and [[ATP synthase]] in the [[phosphorylation system]]. 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 chemiosmotic energy (respiration in heterotrophic cells). ADP is added to [[mitochondrial preparations]] at kinetically saturating concentrations to induce the active state for evaluation of [[OXPHOS capacity]].
 |info=[[MiPNet03.02]], [[MiPNet09.12]]
-|type=Substrate PS
+}}{{MitoPedia topics
-}}
-{{MitoPedia concepts|type=Substrate PS
-}}
-{{MitoPedia methods|type=Substrate PS
-}}
-{{MitoPedia O2k and high-resolution respirometry|type=Substrate PS
-}}
-{{MitoPedia topics
 |mitopedia topic=Substrate and metabolite
-|type=Substrate PS
 }}
 == 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),
 Merck  117105-1GM ('''Calbiochem''') FW = 501.3; store at 4 °C. Recommended due to lower ATP impurities, we use 'ADP-Mg' in [[AT_Innsbruck_OROBOROS |OROBOROS Lab]].
-Alternative source:  Sigma A 5285, 1 g, store at -20 °C; FW = 501.3.
+:::: Alternative source:  Sigma A 5285, 1 g, store at -20 °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>
-'''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) Weigh 501.3 mg of ADP = 1 mmol ADP
+::::# Weigh 501.3 mg of ADP = 1 mmol ADP
-::2) Add 1.2 ml H<sub>2</sub>O  (ADP is not dissolved at this stage).
+::::# Add 1.2 ml H<sub>2</sub>O  (ADP is not dissolved at this stage).
-::3) Neutralize with 5 M KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
+::::# Neutralize with 5 M KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
-::4) Check pH and adjust to 7 if necessary.
+::::# Check pH and adjust to 7 if necessary.
-::5) Adjust final volume to 2 ml and divide into 0.2 ml portions.
+::::# Adjust final volume to 2 ml and divide into 0.2 ml portions.
-::6) Store at -80 °C.
+::::# Store at -80 °C.
-'''Preparation  of 500 mM ADP stock solution with 300 mM free  Mg<sup>2+</sup>''' (dissolved in  H<sub>2</sub>O):
+::: '''Preparation  of 500 mM ADP stock solution with 300 mM free  Mg<sup>2+</sup>''' (dissolved in  H<sub>2</sub>O):
-To  keep free  [Mg<sup>2+</sup>] constant during respiration  measurement  in [[MiR06]] or MiR05, mix ADP with MgCl<sub>2</sub> (0.6 mol   MgCl<sub>2</sub>/mol ADP).
+:::: To  keep free  [Mg<sup>2+</sup>] constant during respiration  measurement  in [[MiR06]] or MiR05, mix ADP with MgCl<sub>2</sub> (0.6 mol   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) Weigh 501.3 mg of ADP = 1 mmol ADP.
+::::# Weigh 501.3 mg of ADP = 1 mmol ADP.
-::2) Add 1.2 ml H<sub>2</sub>O  (ADP is not dissolved at this stage).
+::::# Add 1.2 ml H<sub>2</sub>O  (ADP is not dissolved at this stage).
-::3) Neutralize with 5 M KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
+::::# Neutralize with 5 M KOH (approx. 450 µl). ADP will dissolve after addition of KOH.
-::4)  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.
+::::# 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) Check pH and adjust to 7 if necessary.
+::::# Check pH and adjust to 7 if necessary.
-::6) Adjust final volume to 2 ml and divide into 0.2 ml portions.
+::::# Adjust final volume to 2 ml and divide into 0.2 ml portions.
-::7) Store at -80 °C.
+::::# 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 MgCl<sub>2</sub> solution (0.6 mol MgCl<sub>2</sub>/mol ADP) seperately and perform a titration of both solutions in immediate succession.
+:::: '''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 MgCl<sub>2</sub> solution (0.6 mol MgCl<sub>2</sub>/mol ADP) seperately and perform a titration of both solutions in immediate succession.
-'''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
+::: '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
-::* Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber).
+::::* Titration volume: 4-20 µl using a 25 µl syringe (2 ml O2k-chamber).
-::* Final concentration: 1-5 mM.
+::::* Final concentration: 1-5 mM.