Conformational effects in the high-performance liquid chromatography of proteins. Further studies of the reversed-phase chromatographic behavior of ribonuclease A.
Conformational effects in the high-performance liquid chromatography of proteins. Further studies of the reversed-phase chromatographic behavior of ribonuclease A.
复制标题
蛋白质高效液相色谱中的构象效应。
DOI：
10.1016/0021-9673(86)80059-0
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Karger,BL
中科院分区：
文献类型：
--
作者：
Lu,XM;Benedek,K;Karger,BL
本文是核糖核酸酶A在反相液相色谱中构象效应研究的继续。RNA酶A是色谱洗脱过程中可逆构象重折叠的一个特别好的例子。与光电二极管阵列检测器的吸光度比测量是一致的两个状态的模型，其中早期cluted宽带与折叠或原生状态和后期洗脱的尖锐带与未折叠状态相关联。通过改变移动的流速，同时保持蛋白质在梯度洗脱中的平均容量因子（k′）恒定，测量了移动的相中的复性表观速率常数，发现与文献报道的结果相当。进一步表明，随着梯度陡度参数bor平均值的改变，表观速率常数也发生变化，这是移动的相组成变化的结果。表观速率常数随温度的变化在20°C时达到最大值。基于此模型系统的结果，它是可能的预测的最佳条件下洗脱的物种与尖锐的洗脱峰时，可逆的复性发生在柱中。
This paper is a continuation of an examination of the conformational effects of ribonuclease A (RNase A) in reversed-phase liquid chromatography. RNase A is a particularly good example of reversible conformational refolding during chromatographic elution. Absorbance ratio measurements with a photodiode-array detector are consistent with a two-state model in which an early cluted broad band is associated with the folded or native state and a late eluted sharp band is associated with an unfolded state. By varying the mobile phase flow-rate while maintaining the mean capacity factor (k′) of the protein in gradient elution constant, the apparent rate constant of refolding in the mobile phase was measured and found to be comparable with that reported in the literature. It has been further shown that, as the gradient steepness parameterbor meank′value is attered, the apparent rate constant also changes, as a result of variation in mobile phase composition. The apparent rate constant as a function of temperature was also found to reach a maximum at 20°C. Based on the results of this model system, it is possible to predict optimum conditions for elution of species with sharp elution peaks when reversible refolding takes place in the column.