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Adenosine 5'-triphosphate (ATP)

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.Source: Disodium salt
   Form: Crystalline powder 
   Molecular Weight: 605.1 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20° C (-4° F) 
   Catalog No.: 210J0000 
$15.00/g


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

 

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