Adenosine 5'-triphosphate (ATP) is used in the assay of various ATP dependant enzymes (e.g. hexokinase, phosphoglycerate kinase, acetate kinase) and in the enzymatic determination of many metabolites (e.g. glucose, fructose, mannose, maltose, surcose, triglycerides, creatinine, etc.) ATP is also a component of special pharmaceuticals.

1. 0.1M Triethanolamine buffer/substrate, pH 7.6: 1.86 g TEA¿ HCl, 210 mg glycerate-3-P, 125 mg MgSO₄¿ 7H₂O and 50 mg EDTA with 80 ml distilled water. Adjust pH to 7.6 with 1M NaOH-Na₂ and adjust volume to 100 ml with distilled water.
2. 14 mM NADH: 10 mg NADH-Na₂ with 1 ml distilled water.
3. Glyceraldehyde-3-phosphate dehydrogenase, from rabbit muscle: 10 mg protein/ml (80 U/mg).
4. 3-Phosphoglycerate kinase, from yeast: 10 mg protein/ml (450 U/mg).

1. Dissolve 25 mg ATP 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:

		BLANK	SAMPLE
Buffer/subst.	(1)	2.90 ml	2.90 ml
NADH	(2)	0.05 ml	0.05 ml
sample		- - -	0.10 ml
distilled H2O		0.10 ml	- - -

Mix and read absorbance A₁. 4. To both blank and sample add:

GAP-DH	(3)	0.02 ml	0.02 ml
PGK	(4)	0.02 ml	0.02 ml

Mix and read the absorbance A₂ when the reaction is complete.

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



Total Vol. = 3.09 ml
605.2 = MW of Adenosine 5'-diphosphate
Sample Vol. = 0.10 ml