Ahmad F, Salahuddin A
Biochemistry. 1976 Nov 16;15(23):5168-75. doi: 10.1021/bi00668a034.
The guanidine hydrochloride induced unfolding of the major fraction of ovalbumin (i.e. A1 which contains two phosphate groups and constitutes about 77% of the total protein) was investigated systematically by difference spectran and viscosity measurements. As judged by the intrinsic viscosity (3.9 ml/g), the native protein conformation is compact and globular. Difference spectral results showed extensive disruption of the native structure by guanidine hydrochloride with and without 0.1 M beta-mercaptoethanol were 31.1 and 27.0 ml/g. These and optical rotation results indicated that the denatured protein existed in a cross-linked random coil conformation in 6 M guanidine hydrochloride alone. Strikingly, in contrast to whole ovalbumin, the denaturation of its A1 fraction by guanidine hydrochloride was fully reversible and obeyed first-order kinetic law under different experimental condit ions of pH, temperature, and the denaturant concentration. The monotonic variation of deltaH for the unfolding of ovalbumin A1 by guanidine hydrochloride with temperature, the coincidence of the two transition curves obtained by measuring two independent properties (namely reduced viscosity and difference in light absorption at 288 nm (or 293 nm) as a function of the denaturant concentration, and finally the adherence of the unfolding as well as refolding reactions to first-order kinetic law suggested that the transition of ovalbumin. A1 can reasonably be approximated by a two-state mode. Analysis of the equilibrium data obtained at pH 7.0 and 25 degrees C according to Aune and Tanford (Aune, K.C.,and Tanford, C. (1969), Biochemistry 8, 4586) showed that 12 additional binding sites for the denaturant with an association constant of 1.12 were freshly exposed by the unfolding process and that the native protein was marginally more stable (approximately 6 kcal/mol) than its unfolded form even under native condition. The temperature dependence of the equilibrium constant for the unfolding of ovalbumin A1 by guanidine hydrochloride which was studied in the range 10-60 degrees C at pH 7.0 can be described by assigning the following values of the thermodynamic parameters for the unfolding process: deltaH = 52 kcal/mol at 25 degrees C; deltaS = 153 cal deg-1 mol-1 at 25 degrees C; and delta Cp = 2700 +/- 400 cal deg-1 mol-1.
通过差示光谱和粘度测量系统地研究了盐酸胍诱导的卵清蛋白主要组分（即含有两个磷酸基团且占总蛋白约77%的A1）的去折叠。根据特性粘度（3.9 ml/g）判断，天然蛋白质构象紧密且呈球状。差示光谱结果表明，在有和没有0.1 Mβ-巯基乙醇的情况下，盐酸胍都会使天然结构发生广泛破坏，其特性粘度分别为31.1和27.0 ml/g。这些结果以及旋光结果表明，变性蛋白在单独的6 M盐酸胍中以交联无规卷曲构象存在。引人注目的是，与完整的卵清蛋白不同，盐酸胍对其A1组分的变性是完全可逆的，并且在不同的pH、温度和变性剂浓度实验条件下遵循一级动力学定律。盐酸胍诱导卵清蛋白A1去折叠的ΔH随温度的单调变化、通过测量两个独立性质（即比浓粘度和288 nm（或293 nm）处光吸收差异作为变性剂浓度的函数）获得的两条转变曲线的重合，以及最后去折叠和重折叠反应对一级动力学定律的遵循，表明卵清蛋白A1的转变可以合理地用两态模型近似。根据Aune和Tanford（Aune, K.C., and Tanford, C. (1969), Biochemistry 8, 4586）对在pH 7.0和25℃下获得的平衡数据的分析表明，去折叠过程中新暴露了12个变性剂的额外结合位点，其缔合常数为1.12，并且即使在天然条件下，天然蛋白也比其未折叠形式稍微稳定一些（约6 kcal/mol）。在pH 7.0下于10 - 60℃范围内研究的盐酸胍诱导卵清蛋白A1去折叠的平衡常数的温度依赖性，可以通过为去折叠过程指定以下热力学参数值来描述：25℃时ΔH = 52 kcal/mol；25℃时ΔS = 153 cal deg-1 mol-1；以及ΔCp = 2700 ± 400 cal deg-1 mol-1。
