荧光光谱法研究牛血清蛋白在尿素体系中的变性

Denaturation study of bovine serum albumin induced by the urea by fluorescence spectroscopy

应用荧光光谱技术,对尿素与牛血清蛋白在30℃水溶液中的结合作用及造成牛血清蛋白变性的过程进行了研究,获取了尿素诱导牛血清蛋白变性时相对荧光强度和峰位的变化规律。用Pace等提出的公式分析了相对荧光强度数据,得到了牛血清蛋白变性时的伸展分数fu随溶液pH值和尿素浓度的变化规律。求出了变性平衡常数Ku,伸展吉布斯自由能ΔGu,衡量蛋白质对变性剂稳定性的参量ΔGH2O以及衡量蛋白质变性协同性的参量m和变性中点[D]1/2。通过对实验结果的热力学分析表明,尿素是通过直接和间接的两种作用造成牛血清蛋白变性的,伸展分数fu随尿素浓度的变化呈现3个阶段:尿素浓度达到4 mol.L-1之前为第一阶段,主要产生的是尿素与牛血清蛋白的直接结合作用,在弱酸性条件下这种作用较强;尿素浓度为4 mol.L-1到6 mol.L-1之间为第二阶段,主要通过与溶剂水的作用间接造成蛋白质疏水基团周围水的氢键结构的变化,牛血清蛋白的结构变得更为紧密,形成蛋白质粘稠效应。尿素浓度超过6 mol.L-1之后为第三阶段,尿素分子继续与暴露出来的基团进行结合,当尿素浓度达到8 mol.L-1时,伸展分数约为1,结构完全打开,牛血清蛋白完全变性。

In this paper,the interaction between urea and bovine serum albumin（BSA）in aqueous solution as well as the denaturation process of BSA were studied by using fluorescence spectroscopy technique at 30℃ and three pHs.The fluorescence spectroscopic data have been analysed by using the methods described by Pace.The unfolded fraction（fu）,the denaturation equilibrium constant（Ku）and the free energy of unfolding（△Gu）of protein have been calculated.Then,the transition midpoint（D1/2）and the steepness of the transition region（m）and the free energy of unfolding（ΔGH2O）were obtained.The results which were obtained by thermodynamic analysis indicated that the denaturation of BSA induced by urea is through direct and indirect effects,the interaction between urea and BSA is divided into three stages：the first stage is at the urea concentration below 4 mol·L-1.In this stage,the direct interaction between urea and BSA is the main interaction,while the direct interaction is stronger under acidulous condition.The second stage is in the urea concentrations from 4 mol·L-1 to 6 mol·L-1.In this stage,the structure of the water around the BSA is changed by the interaction of urea and solvent water.BSA structure becomes more closely,that is protein stiffening.The third stage is at the urea concentrations higher than 6 mol·L-1.In this stage,urea molecules combine with the exposed groups.At the urea concentrations is 8mol·L-1,the unfolded fractionfuabout 1,the denaturation of bovine serum albumin induced by urea completely.