Separation of transfer ribonucleic acids by reverse phase chromatography.

Numerous experimental techniques for the separation of transfer ribonucleic acids have been used successfully in preparing partially purified fractions of several specific tRNAs.1 Many of the existing methods have depended upon the differential solubility of specific tRNAs in complex two-phase systems (I-B), and the separations were achieved by means of countercurrent extraction techniques. Column chromatographic experiments with the use of cellulose exchangers (7, 8), methylated albumin (9, lo), or solvent phases supported on inert material (11-13) have also shown partial separation of specific tRNAs. Paper chromatographic procedures have produced partial resolutions of tRNA (14). Methods involving chemical treatment of specific tRNAs have been reported (15-19). Because of our ultimate desire to prepare large quantities of highly purified specific tRNAs, we embarked on a procedure somewhat different from those employed by other investigators. We decided that a minimum number of components and ease of operation should be the primary criteria of our procedure. It seemed likely that column chromatography, combining both ion exchange and differential solubility phenomena, might produce significant resolutions. Furthermore, such a system could be readily adapted to large scale procedures. Our choice of ion exchange agent was the quaternary ammonium compounds because of their demonstrated exchange properties at neutral pH and their solubility in organic solvents. It had been previously observed that quaternary ammonium compounds react with nucleic acids without modification of their activity (20, 21). The organic phase was selected after testing a series of water-insoluble solvents2 for their compatibility with the ion exchange agent. The organic phase was also selected for its ability to extract tRNA from aqueous solutions.