用尺寸排阻色谱法研究蛋白质的热稳定性

Study on the Thermal Stability of Proteins Using Size Exclusion Chromatography

用尺寸排阻色谱Ｚｏｒｂａｘ ＧＦ２５０ 凝胶过滤柱研究了核糖核酸酶Ａ、马心肌红蛋白和溶菌酶的热稳定性。测定了蛋白质在尺寸排阻系统中的分配系数Ｋｄ，并用荧光检测器分析了加热引起的蛋白质内源性荧光的改变，从而计算出了蛋白质的热变性转化点温度。测量了蛋白质天然态、热变性和盐酸胍诱导变性状态下的斯托克斯半径，实验结果进一步证实了热变性后蛋白质不是呈真正的随机线状，而是保留了部分紧密的结构区域。

The thermal stability of ribonuclease A,horse heart myoglobin and lysozyme has been studied using size exclusion chromatography on a Zorbax GF 250 gel permeation column.The thermal unfolding transition temperatures were determined according to the distribution coefficient K d and endogenous fluorescence measuned by a fluorescence detector.The Stokes radii of native,thermal and the guanidinium chloride induced denatured proteins have been stuided,the results provide further evidence for thermal unfolding state of these proteins not being true random coils,but retaining regions of ordered structures.The influence of pH on the thermal stability of ribonuclease A was also examined.