Shastry M C, Eftink M R
Department of Chemistry, University of Mississippi, 38677, USA.
Biochemistry. 1996 Apr 2;35(13):4094-101. doi: 10.1021/bi952550t.
The reverse micellar system formed by the negatively charged surfactant AOT and the organic solvent isooctane is used to solubilize the protein RNase T1. The physicochemical properties of the entrapped protein have been studied using intrinsic tryptophan fluorescence and far-and near-UV CD. These studies indicate a similar structure for the protein in reverse micelles and in pH 7.0 buffer. Thermal unfolding has been studied as a function of W0, the molar ratio of water to AOT, in the solution. Measuring the change in fluorescence intensity as a function of temperature, we observe a reversible transition for W0 in the range 5-12. Heating rate dependencies carried out on these transitions (0.6-3.0 degrees C/min) indicate that the transition temperature and the apparent van't Hoff enthalpy change depend on the scanning rate as well as on W0. The values of the transition temperature, T(m) and the enthalpy change, delta H degrees(un), extrapolated to an infinitely slow scanning rate, are analyzed considering the electrostatic interaction of the charged residues of the protein with the charges of the surfactant molecules forming reverse micelles, the variation of the size of the reverse micelles, and the relative rates of unfolding, refolding, and irreversible denaturation.
由带负电荷的表面活性剂AOT和有机溶剂异辛烷形成的反胶束体系用于溶解蛋白质核糖核酸酶T1。利用内源色氨酸荧光以及远紫外和近紫外圆二色光谱研究了包封蛋白质的物理化学性质。这些研究表明反胶束中的蛋白质与pH 7.0缓冲液中的蛋白质具有相似的结构。研究了热变性与溶液中水与AOT的摩尔比W0的函数关系。通过测量荧光强度随温度的变化，我们观察到W0在5至12范围内的可逆转变。对这些转变进行的升温速率依赖性研究（0.6 - 3.0℃/分钟）表明，转变温度和表观范特霍夫焓变既取决于扫描速率，也取决于W0。考虑到蛋白质带电残基与形成反胶束的表面活性剂分子电荷之间的静电相互作用、反胶束尺寸的变化以及去折叠、重折叠和不可逆变性的相对速率，分析了外推至无限慢扫描速率时的转变温度T(m)和焓变ΔH°(un)的值。
