【摘要】 在15~85℃宽温度范围,研究了蛋白质在固定Zn2+金属螯合色谱系统中的热行为和变性热力学。实验结果表明,蛋白质在色谱过程都有一个固定的热转变温度:核糖核酸酶(RNase)、α-胰凝乳蛋白酶原A(α-Chy)的热转变温度约为55℃,细胞色素C(Cyt-C)和溶菌酶(Lys)约为65℃;,热转变温度的出现标志蛋白质构象发生变化;利用Van′tHoff作图测定了蛋白质在色谱系统热变性时的标准焓变ΔH°和标准熵变ΔS°,提出用标准熵变ΔS°和自由能变ΔG°判断蛋白质构象变化;利用ΔH°-ΔS°的线性关系估算了蛋白质热变性时的补偿温度,鉴定了蛋白质各变体在金属螯合色谱中保留机理的同一性,RNase、Cyt-C、Lys和α-Chy的补偿温度分别为55℃、65.8℃、65.2℃和54.8℃;根据蛋白质热变性时的补偿温度和构象变化熵变Δ(ΔS°)的大小,讨论了蛋白质在阳离子交换色谱和固定Zn的金属螯合色谱体系中的热稳定性。实验证明,在IDA裸柱引入Zn2+后蛋白质在色谱系统中的热稳定性减小,平均补偿温度从65.3℃降低到59.7℃,而构象变化熵变的绝对值大幅度升高。更多还原

【Abstract】 The thermal behavior and denatured thermodynamics of proteins in immobilized Zn²⁺ metal chelate chromatography were investigated in 15⁸5℃.The experimental results show that there is a fixed thermal transition temperature for each protein in chromatographic process.The thermal transition temperature is about 55℃ for ribonuclease（RNase） and chymotrypsinogen A（α-Chy）,and 65℃ for cytochrome C（Cyt-C） and lysozyme（Lys）.The appearance of thermal transition temperature indicates a change of protein conformation.The standard enthalpy change（ΔH°） and entropy change（（ΔS°）） at thermal denaturation of proteins in chromatographic system were determined by using Van′t Hoff relationship（lnk′-（1/T））.It is proposed that the conformation change of protein can be judged by the standard entropy change（ΔS°） and free energy change（ΔG°）.The linear relationship between ΔH°and ΔS°can be used to evaluate the compensation temperature（β） at the protein thermal denaturation and identify the identity of protein retention mechanism in metal（chelate） chromatography.β values of RNase,Cyt-C,Lys and α-Chy are 55℃,65.8℃,65.2℃ and 54.8℃,（respectively）.According to the compensation temperature and the entropy change of conformation change（（Δ（ΔS°）））,the thermostability of proteins was discussed in cation-exchange and immobilized Zn²⁺ metal（chelate） chromatography.The results demonstrate that the thermostability of proteins would be decreased in the chromatographic system after introducing Zn²⁺ into the naked iminodiacetic acid（IDA） column,the average compensation temperature（β） decreases from 65.3℃ to 59.7℃,whereas the absolute value of Δ（ΔS°）（increases） greatly.更多还原