Hydroxyapatite chromatography was first applied to fractionation of tRNA by Hartmann and Coy'. Since then, a number of methodological studies have ap-peared2+, which suggest that this method has a vast potential both for purification of individual species of tRNA and as an analytical tool for tRNA mixtures. Hydroxyapatite chromatography has been successfully applied to the purification of some tRNA species'-r3, although its use since then has been rather restricted. The reason for this is presumably two-fold. First, it is difficult to prepare batches of hydroxyapatite reproducibly. Second, the packing properties of hydroxyapatite columns changed rapidly with time, hence the same column gradually gave irreproducible results. The introduction of agar-entrapped hydroxyapatite has, however, made it possible to use repeatedly the column without change of properties.
The mechanism of interaction between tRNA and hydroxyapatite has not been conclusively elucidated. The work of Bernardi14*' s and others indicates that the essential principle may be electrostatic interaction between calcium ions of the hydroxyapatite and phosphate groups of the tRNA. There is probably also an electrostatic repulsion between the phosphate groups of hydroxyapatite and the phosphodiester groups of the tRNA6. Therefore, the different elution properties of the individual tRNA species reflect the accessibility for electrostatic interaction of the phosphate groups in tRNA.
Any factor that affects the charge of the calcium or phosphate ions of the hydroxyapatite or the charge of steric accessibility of the phosphodiester groups of tRNA should influence the chromatographic pattern. It has been shown that a decrease in pH results in stronger binding of tRNA, possibly owing to decreased protonization of the phosphate groups of hydroxyapatite and hence a weakening of the phosphate-phosphodiester repulsiorP. Also, the kind of positively charged counterions, which more or less neutralize the phosphodiester groups, strongly influences the elution pattern. Thus the binding of tRNA is much weaker in the presence of potassium phosphate buffer than in the presence of sodium phosphate buffer of the same pH and molarity4ss.
Another parameter of interest is temperature. It has been demonstrated with several chromatographic systems utilizing ionic interaction that tRNA is more strongly adsorbed at higher temperatures 16-lg. This effect is ascribed to a loosening