Micromeasurement of nucleoside 5′-triphosphates using coupled bioluminescence

✍ Scribed by Gary A. Goodrich; Harry R. Burrell

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 733 KB
• Volume: 127
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90193-2

✦ Synopsis

Nucleoside S-triphosphates in biological samples can be quantitated accurately to 0.1 pmol using a thin-layer chromatography (TLC)-coupled bioluminescence procedure. 32P-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 Sdiphosphate 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 IO-fold increase in sensitivity as compared to previously described techniques for nucleotide measurement.