Rapid preparation of nucleotides from acid-soluble pools by chromatography on silica, as exemplified with acid extracts of cultured cells.

Abstract

A rapid technique (5-10 min) has been developed for fractionating nucleotides from base and nucleoside contaminants in acid extracts of cells, by adsorption to silica gels. Silica gels (1-mL bed volume) were washed with 5 mL of water then with 5 mL of acetonitrile/water (90/10 by vol). After applying 3-mL samples, adjusted to 900 mL/L acetonitrile content, we washed the gel with an additional 10 mL of the acetonitrile/water solvent. More than 95% of the amounts of bases and nucleosides prsent, except for cytidine (92%), did not adsorb to silica under these conditions. Nucleotides were then quantitatively eluted with 9 mL of water. The retention volumes for positive, negative, and neutral nucleic acid components have been determined, to investigate the discriminatory properties of nucleic acid components on silica. Compounds (bases, nucleosides) that are not ionized at pH 7 do not bind to silica. However, negative, positive, and zwitterionic compounds are tightly adsorbed to the silica gels. This procedure has been used to purify nucleotides from several normal and transformed cell lines.