The assay is based on the reactions described by J.L. Strominger, L.A. Heppel, E.S. Maxwell, Biochim.
Applications
Adenosine 5′-diphosphate (ADP) is used in the determination of creatine kinase, glutamate dehydrogenase, urea and ammonia.
Reagents
Triethanolamine buffer, 0.1M, pH 7.6: 1.86 g TEA• HCl in 80 ml distilled water. Adjust to pH 7.6 with 1.0 M NaOH, adjust volume to 100 ml with distilled water.
14mM NADH:10 mg NADH-Na2 with 1 ml distilled water.
0.5 M MgSO4/2 M KCl: 1.23 g MgSO4• 7H2O and 1.49 g KCl in 10 ml distilled water.
32 mM PEP: 5 mg PEP-(CHA)3 in 1 ml solution (3).
LDH, from rabbit muscle: 5 mg protein/ml (~550 U/mg).
PK, from rabbit muscle: 10 mg protein/ml (200 U/mg).
Procedure
Dissolve 50 mg ADP in 50 ml distilled water in a volumetric flask.
Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25° C.
Into a cuvette, pipette the following:
Buffer (1) 2.70 ml
NADH (2) 0.05 ml
PEP (4) 0.15 ml
LDH (5) 0.02 ml
sample 0.10 ml
Mix and read the absorbance A
Start the reaction by adding 0.02 ml PK (6) and read the absorbance A2.
When the reaction is complete add an additional 0.02 ml PK (6). Mix and read the absorbance A3.
