用尺寸排阻色谱测定蛋白质的构象转化点

建立了用尺寸排阻色谱研究蛋白质构象改变转化点的方法,测定了核糖核酸酶A在尺寸排阻色谱系统中的分配系数Kd,并用荧光检测器监测了加热所导致的蛋白质内源性荧光变化,从而计算出了核糖核酸酶A的热变性转化温度.通过液相色谱梯度洗脱系统在线增加尿素浓度,测定Kd值的动态变化,计算出使溶菌酶发生变性的尿素溶液浓度.用所建立的方法绘制了蛋白质变性前后的尺寸排阻色谱工作曲线,探讨了溶液pH对核糖核酸酶A热稳定性的影响.

Determination of Protein Conformational Transition Points by Size Exclusion Chromatography

A method for the determination of protein conformational transition points was established by size exclusion chromatography.The thermal unfolding transition temperature of ribonuclease A was measured according to the distribution coefficient Kd and the endogenous fluorescence of the chromatographic peak during the heating induced denaturation.The dynamic changes of the lysozyme's Kd was induced by online increasing urea concentration using gradient elution system of the liquid chromatographic system,the urea concentration made the conformational transition of lysozyme was determined by the Kd calculation.The calibration curves for the folding /unfolding proteins in the size exclusion chromatographic system were ploted.The influence of pH on the thermal stability of ribonuclease A was examined.