Haque Inamul, Islam Asimul, Singh Rajendrakumar, Moosavi-Movahedi Ali Akbar, Ahmad Faizan
Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi-110 025, India.
Biophys Chem. 2006 Feb 1;119(3):224-33. doi: 10.1016/j.bpc.2005.09.016. Epub 2005 Oct 14.
We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.e., from their isothermal (25 degrees C) guanidinium chloride (GdmCl)-induced denaturations. We show that our earlier conclusions drawn from heat-induced denaturation studies are correct. Since the extent of unfolding of heat- and GdmCl-induced denatured states of these proteins is not identical, the extent of stabilization of the proteins by polyols against heat and GdmCl denaturations may also differ. We report that in spite of the differences in the structural nature of the heat- and GdmCl-denatured states of each protein, the extent of stabilization by a polyol is same. We also report that the functional dependence of deltaG(D) of proteins in the presence of polyols on denaturant concentration is linear through the full denaturant concentration range. Furthermore, polyols do not affect the secondary and tertiary structures of the native and GdmCl-denatured states.
我们最近从热诱导变性研究中得出结论，在生理pH值和温度下，多元醇不会影响核糖核酸酶A和溶菌酶的ΔG(D)°（25℃时的吉布斯自由能变化（ΔG(D)）），并且它们的稳定作用会随着pH值的降低而增强。由于从热诱导变性曲线估算蛋白质的ΔG(D)°需要进行大量外推，因此该方法估算ΔG(D)°的可靠性一直存在疑问，基于此类研究得出的结论也是如此。这促使我们使用更精确的方法，即通过等温（25℃）氯化胍（GdmCl）诱导变性来测量核糖核酸酶A和溶菌酶的ΔG(D)°。我们发现，我们早期从热诱导变性研究中得出的结论是正确的。由于这些蛋白质的热诱导变性状态和GdmCl诱导变性状态的展开程度并不相同，多元醇对蛋白质抗热变性和抗GdmCl变性的稳定程度可能也有所不同。我们报告称，尽管每种蛋白质的热变性状态和GdmCl变性状态在结构性质上存在差异，但多元醇的稳定程度是相同的。我们还报告称，在多元醇存在的情况下，蛋白质的ΔG(D)对变性剂浓度的功能依赖性在整个变性剂浓度范围内呈线性关系。此外，多元醇不会影响天然状态和GdmCl变性状态的二级和三级结构。
