Labeled adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')tetraphospho(5')nucleoside (Ap4N). Synthesis...

Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(5')tetraphospho(5')adenosine (Ap4A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap4A. [32P]Ap4A has also been synthesized from ATP and [32P]PPi. In a first step, [beta, gamma-32P]ATP is generated in a ATP-[32P]PPi exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap4A is obtained. Radioactive adenosine(5')tetraphospho(5')nucleoside (Ap4N) can also be synthesized from ATP gamma S and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive ApnA and pnA (n > 4) can also be approached with luciferase.