Separation of nucleic acid hydrolysis products, purines, pyrimidines, nucleosides, nucleotides, ribonucleic acid hydrolyzates, and mixtures from nucleotide syntheses by column chromatography on amberlite XAD-4

Abstract

Amberlite XAD-4 resin has been studied as a support for liquid-solid column chromatography. By coating the resin with triethylammonium bicarbonate, a new and unique separation of nucleic acid components has been achieved. Separations are accomplished with a linear gradient of this buffer from 0.1 to 0.4 M. Separation occurs in the following order: inorganic phosphate, purine or pyrimidine bases, 5'-monophosphates, nucleosides and 5'-diphosphates or 5'-triphosphates; the 2'(3')-monophosphates are eluted after either the 5'mono-, di-or triphosphates. The bases and nucleosides are separated in the order: cytosine, uracil, guanine and adenine. Inorganic phosphate and the nucleotides are eluted in the order: inorganic phosphate 5'-mono, di- and tri-phosphates. Excellent separation of the 5'-monophosphates and the 2'(3')-monophosphates is now possible. In each series of 5'-mono-, di- and tri-phosphates or 2'(3')-monophosphates, the elution order is generally cytidine, uridine, guanosine and adenosine. By use of water instead of coating the resin with triethylammonium bicarbonate, the nucleotides and inorganic phosphate are found in the void volume; adenine is eluted very slowly, whereas adenosine is not eluted. Adenosine is eluted only with ethanol-water (1:3). The method is advantageous in that the recovery is quantitative, the buffer is easily removed, the capacity of the column is large (35 mugmoles pergram of resin), flow-rates are high, the time required is short and separations of combinations of inorganic phosphate, bases, nucleosides and nucleotides are now possible that previously could not be accomplished.