Thermal behavior of proteins in high-performance hydrophobic-interaction chromatography. On-line spectroscopic and chromatographic characterization.
Thermal behavior of proteins in high-performance hydrophobic-interaction chromatography. On-line spectroscopic and chromatographic characterization.
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高性能疏水相互作用色谱中蛋白质的热行为。
DOI：
10.1016/0021-9673(86)80058-9
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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：
Wu,SL;Benedek,K;Karger,BL
研究了一系列标准蛋白质在已开发的弱疏水乙醚键合相柱上疏水作用层析的热行为。根据温度和蛋白质的不同，层析体系中可能会发生构象变化。提出了识别这些构象效应的方法，包括保留值和峰宽随温度的变化，以及Z值(logk‘-log%B溶剂曲线的斜率)。Z值是表征蛋白质保留率随盐浓度变化的一般指标。此外，使用光电二极管阵列检测器的在线紫外光谱分析(吸光度比和二阶导数光谱分析)证实了色层分析的趋势。溶菌酶在10-40°C的温度范围内保持稳定的构象，而β-乳球蛋白A在25°C-40°C之间有构象转换，而α-乳清蛋白是一种相当不稳定的物种，它保持稳定的构象直到大约。20°C，然后经历结构变化。最后，细胞色素在整个过程中似乎相对稳定。65°C，因为该蛋白质的构象变化发生在ATCA。对于较疏水的相，固定相的疏水程度对保持天然状态很重要。基于这项工作，亚环境温度下的疏水相互作用层析显示出良好的应用前景。
The thermal behavior of a series of standard proteins in hydrophobic interaction chromatography on a previously developed weakly hydrophobic ether-bonded phase column has been studied. Depending on the temperature and protein, conformational changes can occur in the chromatographic system. Methods for recognizing these conformational effects are presented, including retention and peak width variations with temperature, andZvalues (the slope of the plot of logk′ vs. log %Bsolvent. TheZvalue is shown to be a general index characterizing protein retention as a function of salt concentration. In addition, on-line UV spectroscopic analysis, (absorbance ratios and second derivative spectroscopy) with a photodiode array detector, is shown to corroborate chromatographic trends. Lysozyme maintains a stable conformation over the temperature range 10–40°C, whereas β-lactoglobulin A has a conformational transition between 25°C and 40°C. Calcium-depleted α-lactalbumin, a rather labile species, maintains a stable conformation up toca. 20°C, and then undergoes structural changes. Finally, cytochromecappears to be relatively stable up toca. 65°C. Since conformational changes for this protein occur atca. 35°C on more hydrophobic phases, the extent of hydrophobicity of the stationary phase is important for maintenance of the native state. Based on this work, hydrophobic-interaction chromatography at sub-ambient temperatures appears promising.