Micromeasurement of nucleoside 5′-triphosphates using coupled bioluminescence

Nucleoside 5′-triphosphates in biological samples can be quantitated accurately to 0.1 pmol using a thin-layer chromatography (TLC)-coupled bioluminescence procedure. 32 P-Labeled nucleotides of known specific activities are added to a cell extract as internal standards and the mixture is separated by two-dimensional TLC. Isolated nucleotides are located on the TLC by autoradiography, eluted, and measured in a two-step reaction using nucleoside 5′-diphosphate kinase to catalyze an equimolar phosphorylation of ADP; the mass of resulting ATP is then determined in the firefly luciferin-luciferase reaction. This method provides a 10-fold increase in sensitivity as compared to previously described techniques for nucleotide measurement.