Adenosine 5'-monophosphate (AMP) is used in the determination of creatine kinase and 5'-nucleotidase.

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. 16.5 mM ATP: 10 mg NADH-Na₂ in 1 ml distilled water.
4. 0.5 M MgSO₄/2 M KCl: 1.23 g MgSO₄• 7 H₂O and 1.49 g KCl in 10 ml distilled water.
5. 32 mM Phosphoenolpyruvate: 15 mg PEP-(CHA)₃ in 1 ml MgSO₄/KCl.
6. LDH, from rabbit muscle: 5 mg protein/ml (550 U/mg).
7. Pyruvate kinase, from rabbit muscle: 10 mg protein/ml (200 U/mg).
8. Myokinase, from rabbit muscle: 5 mg protein/ml (360 U/mg).

1. Dissolve 50 mg AMP 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 two cuvettes, pipette the following:

		BLANK	SAMPLE
Buffer	(1)	3.00 ml	3.00 ml
NADH	(2)	0.05 ml	0.05 ml
ATP	(3)	0.01 ml	0.01 ml
PEP	(5)	0.15 ml	0.15 ml
LDH	(6)	0.01 ml	0.10 ml
PK	(7)	0.02 ml	0.02 ml
Distilled H2O		0.05 ml	- - -
Sample		- - -	0.05 ml

Mix and read the absorbance A₁.
4. Start the reaction by adding 0.01 ml myokinase (8) to both the blank and sample. Read the absorbance A₂.

A = (A₁ - A₂ )_sample - (A₁ - A₂ )_blank


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