Mersol J V, Steel D G, Gafni A
Department of Physics, Institute of Gerontology, University of Michigan, Ann Arbor 48109-2007.
Biophys Chem. 1993 Dec;48(2):281-91. doi: 10.1016/0301-4622(93)85015-a.
The reversible denaturation of Escherichia coli alkaline phosphatase (AP) was followed by monitoring changes in enzymatic activity as well as by measurements of the time-resolved room temperature phosphorescence from Trp 109. It is well known that the denaturants, ethylene diamine tetraacetic acid (EDTA), acid and guanidine hydrochloride (GdnHCl) inactive AP by different mechanisms as reflected by differences in the time dependence of inactivation. However, further information about structural changes that result during inactivation is obtained by measurement of the phosphorescence intensity and radiative decay rate. Time-resolved tryptophan phosphorescence is exquisitely sensitive to changes in the local environment of the emitting residue, unlike the steady state phosphorescence intensity which is a composite of both the lifetime and concentration of the emitting protein species. The results show that while inactivation in EDTA proceeds by loss of the zinc ion as expected, denaturation in acid or GdnHCl produces a heterogeneous population of AP molecules, detected by a distribution analysis of the phosphorescence lifetime, which may reflect multiple pathways to the final unfolded state. Time-resolved phosphorescence also demonstrates the existence of an enzymatically active but structurally less rigid intermediate state during unfolding. As the rigidity decreases, the susceptibility to further denaturation decreases at lower pH but increases with GdnHCl concentration. The experiments provide new insight into the mechanism of denaturation of AP and demonstrate the sensitivity of time-resolved room temperature phosphorescence to the structural details of intermediate states produced during unfolding of proteins.
通过监测酶活性的变化以及测量色氨酸109的时间分辨室温磷光，研究了大肠杆菌碱性磷酸酶（AP）的可逆变性。众所周知，变性剂乙二胺四乙酸（EDTA）、酸和盐酸胍（GdnHCl）通过不同机制使AP失活，这反映在失活的时间依赖性差异上。然而，通过测量磷光强度和辐射衰减率，可以获得有关失活过程中结构变化的更多信息。与稳态磷光强度不同，时间分辨色氨酸磷光对发射残基局部环境的变化极为敏感，稳态磷光强度是发射蛋白种类的寿命和浓度的综合体现。结果表明，正如预期的那样，EDTA中的失活是由于锌离子的丢失，而酸或GdnHCl中的变性会产生异质性的AP分子群体，通过磷光寿命的分布分析检测到，这可能反映了通向最终未折叠状态的多种途径。时间分辨磷光还证明了在去折叠过程中存在一种酶活性但结构刚性较低的中间状态。随着刚性降低，在较低pH值下进一步变性的敏感性降低，但随着GdnHCl浓度的增加而增加。这些实验为AP的变性机制提供了新的见解，并证明了时间分辨室温磷光对蛋白质去折叠过程中产生的中间状态结构细节的敏感性。
