France R M, Grossman S H
Department of Chemistry, University of South Florida, Tampa 33620, USA.
Arch Biochem Biophys. 1996 Feb 1;326(1):93-9. doi: 10.1006/abbi.1996.0051.
The unfolding transition of monomeric arginine kinase from shrimp was examined in a multiparameter equilibrium approach. Parameters investigated included catalytic activity, circular dichroism, intrinsic fluorescence characteristics including acrylamide quenching, and steady-state anisotropy of arginine kinase derivatized at the reactive cysteine with fluorescent dye 5-[[[(iodoacetyl)amine]ethyl]amino]-naphthalene-1-sulfonic acid. The time course of electrophoretic patterns in urea gradient gels was also determined. Midpoints of the transitions varied considerably depending upon the parameter, indicating the presence of populated intermediates. Significant unfolding began after 2 M urea with most secondary and tertiary structure eliminated in 5 M urea. In dilute denaturant, arginine kinase exhibited a small increase in specific activity and physical properties characteristic of a protein with collapsed structure, including an increase in alpha-helical content, a decrease in intrinsic fluorescence (without a shift in the emission maximum), an increase in anisotropy, and a decrease in fractional accessibility by tryptophan to acrylamide quenching. The electrophoretic pattern of arginine kinase in urea gradient gels is consistent with the presence of a compact conformation in dilute denaturant. The results indicate the existence of a contracted overall conformation in dilute urea. The persistence of catalytic activity suggests this structure may be a functional molecular isoform, but the obvious differences in structure between the native state and the conformation of arginine kinase in 0.5 M urea raise the question of whether such isoforms may also be a type of folding intermediate.
采用多参数平衡方法研究了虾单体精氨酸激酶的去折叠转变。研究的参数包括催化活性、圆二色性、包括丙烯酰胺猝灭在内的内在荧光特性，以及用荧光染料5-[[[(碘乙酰基)胺]乙基]氨基]-萘-1-磺酸衍生化的精氨酸激酶在反应性半胱氨酸处的稳态各向异性。还测定了尿素梯度凝胶中电泳图谱的时间进程。转变的中点根据参数的不同有很大差异，表明存在大量中间体。在2M尿素后开始显著去折叠，在5M尿素中大部分二级和三级结构被消除。在稀变性剂中，精氨酸激酶表现出比活性略有增加以及具有塌陷结构的蛋白质的物理性质特征，包括α-螺旋含量增加、内在荧光降低（发射最大值无位移）、各向异性增加以及色氨酸对丙烯酰胺猝灭的可及分数降低。尿素梯度凝胶中精氨酸激酶的电泳图谱与稀变性剂中存在紧密构象一致。结果表明在稀尿素中存在收缩的整体构象。催化活性的持续存在表明这种结构可能是一种功能性分子异构体，但天然状态与0.5M尿素中精氨酸激酶构象之间明显的结构差异引发了这样的异构体是否也可能是一种折叠中间体的问题。
