Reaction #2200612

ord-febe6175bf6f43a2bb08ffed752f9dbc

Reaction equation

Nc1ncnc2c1ncn2[C@@H]1O[C@@H]2COP(=O)(O)O[C@H]2[C@H]1O
cAMP
CC(C)C[C@H](NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](N)CC(C)C)C(=O)NCC(=O)O.O=C(O)c1ccccc1-c1c2ccc(=O)cc-2oc2cc(O)ccc12
fluorescein Kemptide
[Cl-].[Cl-].[Mg+2]
MgCl2
O[C@H](CS)[C@H](O)CS
DTT
CC(C)C[C@H](NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](N)CC(C)C)C(=O)NCC(=O)O
Kemptide
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ATP
→
O=C(O)c1ccccc1-c1c2ccc(=O)cc-2oc2cc(O)ccc12
Fluorescein

Reactants

Nc1ncnc2c1ncn2[C@@H]1O[C@@H]2COP(=O)(O)O[C@H]2[C@H]1O
cAMP
CC(C)C[C@H](NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](N)CC(C)C)C(=O)NCC(=O)O.O=C(O)c1ccccc1-c1c2ccc(=O)cc-2oc2cc(O)ccc12
fluorescein Kemptide
[Cl-].[Cl-].[Mg+2]
MgCl2
O[C@H](CS)[C@H](O)CS
DTT
CC(C)C[C@H](NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](N)CC(C)C)C(=O)NCC(=O)O
Kemptide
—
71.25
—
0.25
—
Fe(III)
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ATP

Reagents

None

Solvents

O=S(=O)(O)CCN1CCN(CCO)CC1
HEPES
O=S(=O)(O)CCN1CCN(CCO)CC1
HEPES

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    Otherreaction
  2. 2
    Otherrespectively, with a final reaction volume of 30 ml
  3. 3
    Otherfor a reaction time of 1 hour
  4. 4
    workup.ADDITIONwas added to the enzymatic reaction mixture
  5. 5
    Filtrationfilter

Procedure

PKA was diluted in 20 mM HEPES, pH 7.4, w/0.05% TX-100 and incubated in a final reaction buffer consisting of 20 mM HEPES, pH 7.4, 0.1 mM cAMP, 1 mM ATP, 5 mM MgCl2, 1 mM DTT with either fluorescein Kemptide (71.25 uM) or OREGON GREEN® Kemptide as the enzymatic substrate at a final concentration of 71.25 or 114.25 mM, respectively, with a final reaction volume of 30 ml, for a reaction time of 1 hour in a 96 well plate format using an opaque white plate. Subsequently, 120 ml of 0.25× Fe(III) working reagent prepared according to Example I was added to the enzymatic reaction mixture and the fluorescence was measured using a 485/538 ex/em filter set. The results are shown in FIGS. 14 and 15.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US07432070B2uspto-grants-2008_10