Gupta R, Ahmad F
Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India.
Biochemistry. 1999 Feb 23;38(8):2471-9. doi: 10.1021/bi982078m.
Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.
根据化学变性剂诱导的构象转变曲线来确定蛋白质稳定性（ΔG°D）存在问题；对于不同的ΔG°D值，当使用不同的外推方法分析同一组（ΔGD，变性剂浓度）数据时，可得到在不存在变性剂时变性过程中的吉布斯自由能变化值（ΔGD）[佩斯，C. N.（1986年）《酶学方法》131，266 - 280]。我们提出了这个问题的一个实际解决方案，并将其用于测试溶菌酶、核糖核酸酶 - A和细胞色素 - c的ΔGD对[尿素]（尿素的摩尔浓度）的依赖性。该方法采用：（i）在不同盐酸胍（GdnHCl）浓度存在下测量尿素诱导的变性，在相同温度和pH下，GdnHCl自身会破坏蛋白质的天然状态，此时已测量了尿素和GdnHCl诱导的变性；（ii）估计ΔGDcor，即使用关系式（ΔGDcor = ΔGD + mg [GdnHCl] = ΔG°D - μ [尿素]，其中mg和μ分别是ΔGD对[GdnHCl]和[尿素]的依赖性）对GdnHCl对尿素诱导变性的影响进行校正后的ΔGD值，其参数均由实验变性数据确定；（iii）将ΔGDcor映射到在不存在GdnHCl时获得的ΔGD与[尿素]的关系图上。我们的结果令人信服地表明：（i）每种蛋白质的ΔGD对[尿素]的依赖性在整个浓度范围内是线性的；（ii）尿素和GdnHCl对蛋白质变性的影响是相加的；（iii）如果天然蛋白质含有电荷 - 电荷相互作用和/或阴离子结合位点，KCl会以与晶体结构数据一致的方式影响尿素诱导的变性。
