Synthesis and affinity purification of beta-32P-labeled [gamma-S]GTP

A method for the synthesis and purification of guanosine 5'-gamma-S]triphosphate labeled with 32P in the beta-position is described. The first step in the synthesis involves the quantitative transfer of 32Pi from gamma-32P]dATP to 5'-GMP catalyzed by GMP kinase. Further incubation of the beta-32P]GDP product with gamma-S]GTP and nucleoside diphosphate kinase results in the synthesis of beta-32P]gamma-S]GTP with a yield of 10 to 18%. The 32P-labeled gamma-S]nucleotide is purified by binding to mercury-agarose and eluting with buffer containing beta-mercaptoethanol. Specific incorporation of 32P into the beta-position was demonstrated by treating beta-32P]gamma-S]GTP with 7% formic acid to remove the gamma-thiophosphate and digesting the remaining beta-32P]GDP with nucleotide pyro-phosphatase. Although 5'-GMP was released after pyrophosphatase digestion, the only 32P radioactivity detected was as inorganic phosphate.