Thermal chromatography of lysine-specific transfer ribonucleic acid from Escherichia coli B

Abstract

Two Escherichia coli B tRNALys isoacceptors have been separated by thermal chromatography on hydroxyapatite demonstrating the potential of this approach in the fractionation of single-stranded polynucleotides. Elution temperatures were reproducible and depended primarily on the cation present and its concentration. Cesium phosphate was a more effective eluent than sodium phosphate. The integrity of the polynucleotide following thermal chromatography was examined by ultracentrifugation in a 60% dimethyl sulfoxide solvent, electrophoresis on polyacrylamide gels containing urea and oligonucleotide mapping. Adsorption to hydroxyapatite did not noticeably increase phosphodiester bond breakage compared with that in solution. Minimal, if any, rupture of covalent linkages was detected under the conditions of preparative separation of the isoacceptors. Several observations suggest that only one tRNALys was transcribed by the E. coli B cell.