Investigation of tritium tracer in nucleic acid components by hydrogenolysis of corresponding precursors

Preparation of [5,6-3H] uracil by hydrogenolysis of 5-bromo-6-chlorouracil with molar activity (1.59-1.63)x1015 Bg/mol has been described. Hydrogenolysis reaction kinetics is studied, effect of the reaction conditions on molar activity of dehalogenation product is considered. Using enzyme fraction, separated from the E.coli cells tritium-labelled preparations of UMP, UTP and UTP from [5.6-3H] uracil are made. Kinetics of enzymatic reactions is studied and conditions, permitting to synthesize both UMP and UTP and a mixture of nucleotides of different degree of phosphorylation in approximately equal amounts are selected. Separation of nucleotides labelled with tritium is conducted using ion exchange chromatography on DEAE cellulose. Molar activity of nucleotides labelled with tritium equals molar activity of initial [5.6-3H] uracil, and radiochemical purity constitutes more than 95%