Adenosine 5'-diphosphate (ADP) is used in the determination of creatine kinase, glutamate dehydrogenase, urea and ammonia.

The assay is based on the reactions described by J.L. Strominger, L.A. Heppel, E.S. Maxwell, Biochim. Biophys. Acta., 32, 412, 1956.

1. 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.
2. 14mM NADH:10 mg NADH-Na₂ with 1 ml distilled water.
3. 0.5 M MgSO₄/2 M KCl: 1.23 g MgSO₄• 7H₂O and 1.49 g KCl in 10 ml distilled water.
4. 32 mM PEP: 5 mg PEP-(CHA)₃ in 1 ml solution (3).
5. LDH, from rabbit muscle: 5 mg protein/ml (~550 U/mg).
6. PK, from rabbit muscle: 10 mg protein/ml (200 U/mg).

1. Dissolve 50 mg ADP in 50 ml distilled water in a volumetric flask.
2. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25° C.
3. 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
4. Start the reaction by adding 0.02 ml PK (6) and read the absorbance A₂.
5. When the reaction is complete add an additional 0.02 ml PK (6). Mix and read the absorbance A₃.

A = (A₁ - A₂ ) - (A₂ - A₃ )


Total Vol. = 3.04 ml
449.2 = MW of Adenosine 5'-diphosphate
Sample Vol. = 0.10 ml