Yamakawa Y, Miyasaka K, Ishikawa T, Yamada Y, Okuyama T
Department of Applied Immunology, National Institute of Health, Tokyo, Japan.
J Chromatogr. 1990 May 11;506:319-26. doi: 10.1016/s0021-9673(01)91587-0.
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.
高效液相色谱法（HPLC）被应用于新开发的球形羟基磷灰石珠上，用于分析纯化的大肠杆菌tRNA（缬氨酸、甲硫氨酸、酪氨酸和苯丙氨酸）。通过浓度从64到123 mM的磷酸盐缓冲液（pH 6.8）进行45分钟的梯度洗脱，tRNA从柱中分别洗脱，保留时间存在明显差异；各个tRNA的保留时间和峰面积都具有高度的可重复性。从大肠杆菌和枯草芽孢杆菌获得的总tRNA（tRNA（Total））制剂也在该柱上进行了分析。即使是通常可能包含60种或更多tRNA种类的tRNA（Total），也可以用相对较浅的梯度（如75 - 132 mM）洗脱。tRNA从柱中的回收率高达90%。由于组成复杂，tRNA（Total）的洗脱图谱呈广泛分布的不规则形状，但在1小时的梯度下，很容易检测到十多个峰。当测定tRNA（Total）洗脱液中tRNA的氨基酸接受活性时，对于十种特定的tRNA，每个活性峰都从柱中急剧洗脱。此外，几种tRNA活性在不同的馏分中洗脱。这表明同功受体被该柱分离。结果表明，羟基磷灰石珠上的HPLC可用于tRNA的纯化和表征。
