摘要
考察了一系列标准蛋白质在疏水色谱法中的热行为；研究了柱温变化对蛋白质和小分子保留值的影响。实验结果表明，每个蛋白质的保留值－温度曲线上都有一个固定的热变性转变温度。在这个温度以下，除热稳定性较高的溶菌酶外，蛋白质的保留值都随柱温的升高而线性地增加。在转变温度以上，保留值随柱温升高会迅速增大或减小。与蛋白质相反，小分子的保留值随柱温的升高会不断地减小。研究了柱温变化对酶活性的影响，并与酶的静态热失活进行了比较。发现仅在室温下疏水色谱法才具有温和的分离条件，在较高柱温下疏水固定相可以诱导蛋白质变性。柱温对蛋白质活性的影响取决于固定相疏水性的强弱。通常，在给定温度下疏水性越强，酶越容易失活。测定了不同柱温下α－淀粉酶在疏水色谱法中的Ｚ值（ｌｇｋ′对作图的斜率）。分析了较高柱温下导致蛋白质保留值的增长是由于蛋白质的构象变化。
he thermal behaviors of a series of standard proteins in hydrophobic interaction chromatography have been examined. The influence of column temperature on retentions for proteins and small molecules was investigated. The results showed that there is a fixed thermal denaturation transition temperature in retentiontemperature curve for each protein. Below this temperature, the proteins display a linear increase in retentionwith increasing temperature except lysozyme with higher thermal stability. Over the temperature, the retention of proteins increases or decreases rapidly with column temperature. The retention for small molecule, in contrast to proteins, decreases continually with increasing temperature. The effect of column temperature on enzyme activity was also studied. In comparing hydrophobic interaction chromatography conditions with the static thermal inactivation of the enzyme it was found that a mild separation condition appears only at ambient temperature,while the hydrophobic stationary phase can induce the denaturation of protein under higher temperature. The effect of temperature on enzymatic activity is dependent on the extent of hydrophobicity in the stationary phase. Z values (the slope of the plot of lgk′vs.lg1/B)of α-amylase in hydrophobic interaction chromatography were measured by isocratic elution under different column temperatures. The reason leading to the increase of protein retention under higher temperature was discussed. The results suggest that the proteins undergo conformational changes.
出处
《色谱》
CAS
CSCD
北大核心
1996年第3期172-175,共4页
Chinese Journal of Chromatography
关键词
蛋白质
柱温影响
疏水色谱
hydrophobic-interaction chromatography ,proteins, effect of column temperature
