A modification to an enzymatic method for the preparation of (α-³²P) ATP with a high specific activity

Abstract

A modification of the procedure reported byJohnson andWalseth ¹ for the preparation of (α-³²P)ATP:(γ-³²P)GTP with a specific activity of more than 5000 Ci/mmol was made firstly, then it was used as the phosphate donor in the reaction catalyzed by the polynucleotide kinase, to transfer its radioactive phosphate to the 5′-position of the 3′-AMP, resulting (5′-³²P)3′-ADP. After that, (5′-³²P)3′-ADP was hydrolysed to (5′-³²P)AMP under the catalysis of Nuclease P1, the late was then phosphorylated to produce (α-³²P)ATP by the action of myokinase and pyruvate kinase. By this method, we firstly prepared (α-³²P)ATP with a specific activity of more than 4000 Ci/mmol which was measured by HPLC analysis.