Reverse salt gradient chromatography of tRNA on unsubstituted agarose. II. Further study of binding mechanisms

The existence of two mechanisms in the fractionation of tRNA on Sepharose 4B, using reverse gradients of ammonium sulphate, was confirmed by comparing yeast and E. coli tRNAs, and by extending the range of conditions used. The difference in elution range between the two kinds of tRNA is attributable mainly to variations in tight binding rather than adsorptive retardation. Variations of pH or of temperature produce not only changes in peak distribution, but overall shifts in the elution profile. The shifts correlate closely with changes in the solubility of tRNA in free solution, confirming that tight binding is associated with interfacial precipitation. On the other hand the degree of adsorptive retardation, as indicated by the difference between elution profiles from long and short columns, is quite similar under all conditions. It is if anything slightly greater at 25 degrees C than at 5 degrees C, implying a binding mechanism analogous to hydrophobic bonding. Binding-equilibrium studies suggest that the effect is related to the formation of a monolayer of tRNA on the agarose threads of matrix. Experiments with hydroxyapatite also demonstrate adsorptive retardation, comparable in degree to that observed with Sepharose. This indicates that the effect may be much more important in ion-exchange chromatography than is normally assumed.