Ibarra-Molero B, Sanchez-Ruiz J M
Departamento de Quimica Fisica (Facultad de Ciencias), Instituto de Biotecnologia, Granada, Spain.
Biochemistry. 1996 Nov 26;35(47):14689-702. doi: 10.1021/bi961836a.
We have characterized the guanidine-induced denaturation of hen egg white lysozyme within the 30-75 degrees C temperature range on the basis of equilibrium fluorescence measurements, unfolding assays, kinetic fluorescence measurements, and differential scanning calorimetry. Analysis of the guanidine denaturation profiles according to the linear extrapolation method yields values for the denaturation Gibbs energy which are about 15 kJ/mol lower than those derived from differential scanning calorimetry. Our results strongly suggest that this discrepancy is not due to deviations from the two-state denaturation mechanism. We propose a new method for the determination of denaturation Gibbs energies from solvent-denaturation data (the constant-delta G extrapolation procedure). It employs several solvent-denaturation profiles (obtained at different temperatures) to generate the protein stability curve at zero denaturant concentration within the -8 to 8 kJ/mol delta G range. The method is model-independent and provides a practical, nonlinear alternative to the commonly employed linear extrapolation procedure. The application of the constant-delta G method to our data suggests that the guanidine-concentration dependence of the denaturation Gibbs energy is approximately linear over an extended concentration range but, also, that strong deviations from linearity may occur at low guanidine concentrations. We tentatively attribute these deviations to the abrupt change of the contribution to protein stability that arises from pairwise charge-charge electrostatic interactions. This contribution may be positive, negative, or close to zero, depending on the pH value and the charge distribution on the native protein surface [Yang, A.-S., & Honig, B. (1993) J. Mol. Biol. 231, 459-474], which may help to explain why disparate effects have been found when studying protein denaturation at low guanidine concentrations. Kinetic m values for lysozyme denaturation depend on temperature, in a manner which appears consistent with Hammond behavior.
我们基于平衡荧光测量、去折叠分析、动力学荧光测量和差示扫描量热法，对30至75摄氏度温度范围内胍诱导的鸡蛋清溶菌酶变性进行了表征。根据线性外推法对胍变性曲线进行分析，得到的变性吉布斯自由能值比差示扫描量热法得到的值低约15 kJ/mol。我们的结果强烈表明，这种差异并非由于偏离了两态变性机制。我们提出了一种从溶剂变性数据中测定变性吉布斯自由能的新方法（恒定ΔG外推法）。它采用多个溶剂变性曲线（在不同温度下获得）来生成零变性剂浓度下-8至8 kJ/mol ΔG范围内的蛋白质稳定性曲线。该方法与模型无关，为常用的线性外推法提供了一种实用的非线性替代方法。将恒定ΔG方法应用于我们的数据表明，变性吉布斯自由能对胍浓度的依赖性在扩展的浓度范围内近似线性，但在低胍浓度下也可能出现强烈的线性偏差。我们初步将这些偏差归因于成对电荷 - 电荷静电相互作用对蛋白质稳定性贡献的突然变化。这种贡献可能为正、负或接近零，这取决于pH值和天然蛋白质表面的电荷分布[Yang, A.-S., & Honig, B. (1993) J. Mol. Biol. 231, 459 - 474]，这可能有助于解释为什么在研究低胍浓度下的蛋白质变性时会发现不同的影响。溶菌酶变性的动力学m值取决于温度，其方式似乎与哈蒙德行为一致。
