Spencer M
J Chromatogr. 1982 Apr 23;238(2):307-16. doi: 10.1016/s0021-9673(00)81316-3.
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.
通过比较酵母和大肠杆菌的tRNA，并扩展所用条件的范围，证实了在使用硫酸铵反向梯度的情况下，tRNA在琼脂糖4B上分级分离存在两种机制。两种tRNA洗脱范围的差异主要归因于紧密结合的变化，而非吸附阻滞。pH值或温度的变化不仅会导致峰分布的改变，还会使洗脱曲线整体发生移动。这些移动与tRNA在自由溶液中溶解度的变化密切相关，证实紧密结合与界面沉淀有关。另一方面，从长柱和短柱洗脱曲线的差异所表明的吸附阻滞程度，在所有条件下都非常相似。实际上，在25℃时比在5℃时略大，这意味着存在一种类似于疏水键合的结合机制。结合平衡研究表明，这种效应与tRNA在基质琼脂糖丝上形成单分子层有关。用羟基磷灰石进行的实验也证明了吸附阻滞，其程度与琼脂糖观察到的相当。这表明该效应在离子交换色谱中可能比通常认为的更为重要。
