High-performance liquid chromatography of transfer ribonucleic acids on spherical hydroxyapatite beads

High-performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite was applied to the analysis of purified E. coli tRNAs (Val, Met, Tyr and Phe). tRNAs were eluted from the column separately with appreciable differences in retention time by a 45-min gradient of phosphate buffer (pH 6.8) of concentration from 64 to 123 mM; both the retention times and peak areas of respective tRNAs were highly reproducible. Total tRNA (tRNA(Total)) preparations obtained from E. coli and B. subtilis were also analysed on the column. It is possible even to elute tRNA(Total) which may, in general, contain 60 or more tRNA species with a relatively shallow gradient such as 75-132 mM. The recovery of tRNA from the column was as high as 90%. Owing to the complicated composition, the elution profile of tRNA(Total) had a wide spread irregular shape but, with a 1-h gradient more than ten peaks were easily detected. When the amino acid-accepting activity of tRNA in the eluate of tRNA(Total) was determined, for ten specific tRNAs, each activity peak was eluted sharply from the column. In addition, several tRNA activities were eluted in different fractions. This indicates that isoacceptors were separated by the column. The results show that HPLC on hydroxyapatite beads is useful for the purification and characterization of tRNA.