尿素诱导牛血红蛋白变性的微量热和平衡渗析研究

应用恒温微量热技术和平衡渗析技术,对尿素与牛血红蛋白在30℃水溶液中的结合作用及造成牛血红蛋白变性的过程进行了研究,得到了尿素在牛血红蛋白上的平均结合数、二者之间的结合焓及每摩尔尿素与牛血红蛋白结合的焓效应,并根据简单结合模型,计算了它们之间的结合常数、结合自由能.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红蛋白变性的,它们之间的相互作用分为三个阶段:尿素浓度达到4molL-1之前为第一阶段,主要产生的是尿素与牛血红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4molL-1到6molL-1之间为第二阶段,主要通过尿素与溶剂水的作用造成牛血红蛋白周围水结构的变化,在尿素浓度达到6molL-1时两种作用的叠加造成牛血红蛋白结构的破坏,暴露出原来处于结构内部的基团;尿素浓度超过6molL-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.牛血红蛋白次级结构的维系具有协作性,其结构的破坏不是渐进的,而是一次性全部打开,结构破坏产生的热效应并不太大.

Denaturation study of bovine hemoglobin induced by the urea by microcalorimetry and equilibrium dialysis

The interaction between urea and bovine hemoglobin （BHb） in aqueous solution as well as the denaturation process of BHb were studied by using isothermal microcalorimetry method and equilibrium dialysis technique at 30 ℃ and three pHs.The average binding numbers,the values of binding enthalpy of urea to BHb and the values of enthalpy per mole of urea were obtained.The simple binding model was employed to obtain the binding constant and the binding free energies.The results which were obtained by thermodynamic analysis indicated that the denaturation of BHb induced by urea is through direct and indirect effects,the interaction between urea and BHb 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 BHb 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 BHb is changed by the interaction of urea and solvent water.When the urea concentration reaches 6mol L-1,the two interactions together cause the structural damage of the BHb and expose the groups which are in the inner structure of BHb.The third stage is at the urea concentrations higher than 6 mol L-1.In this stage,urea molecules combine with the exposed groups.The destruction of the structure of BHb is not a gradual process,but rather a completely destruction at once and the thermal effect resulting from the structural damage is not too much.