Resolution of multiphasic reactions by the combination of fluorescence total-intensity and anisotropy stopped-flow kinetic experiments.
Resolution of multiphasic reactions by the combination of fluorescence total-intensity and anisotropy stopped-flow kinetic experiments.
复制标题
通过荧光总强度和各向异性停流动力学实验的结合来解决多相反应。
DOI：
10.1016/s0006-3495(94)80741-6
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发表时间：
1994
影响因子：
3.4
通讯作者：
Beechem,JM
中科院分区：
文献类型：
--
作者：
Otto,MR;Lillo,MP;Beechem,JM
在停流研究中经常观察到多相动力学。为了进一步表征这些动力学阶段，我们开发了一种方法，使荧光总强度和各向异性停流数据可以结合在一个单一的分析。荧光总强度和各向异性是高度相关的，并且包含两种非常互补的信息形式。总强度的变化是有用的，在确定不同的量子产率的人口的变化，而各向异性的变化包含额外的贡献所造成的旋转动力学的物种。对于荧光量子产率增加的情况，观察到的各向异性变化率将比总强度变化更快，而在总强度降低的情况下，观察到的各向异性变化将滞后。在所有情况下，随着量子产率的变化，停流各向异性信号不能与由指数组成的模型拟合。研究这些影响的案例研究描述了葡萄球菌核酸酶和磷酸甘油酸激酶的蛋白质折叠/重折叠过渡。还检查了使用噬菌体T4 DNA聚合酶的多相DNA核酸外切酶反应。在所有这些情况下，两种数据类型的组合分析揭示了对反应机制的见解，这不能通过单独的数据类型获得。量子产率和稳态各向异性与瞬态填充中间物种可以解决。所描述的数据分析方法允许在内部一致的动力学速率、量子产率和稳态各向异性方面表征多相反应。
Multiphasic kinetics are often observed in stopped-flow investigations. To characterize further these kinetic phases, we have developed a methodology whereby fluorescence total intensity and anisotropy stopped-flow data can be combined in a single analysis. Fluorescence total intensity and anisotropy are highly interrelated and contain two very complementary forms of information. Total-intensity changes are useful in determining changes in populations with differing quantum yields, whereas anisotropy changes contain additional contributions caused by the rotational dynamics of the species. For cases in which the fluorescence quantum yield increases, the observed rate of anisotropy change will be more rapid than the total-intensity change, whereas in cases in which the total intensity decreases, the observed change in anisotropy will lag behind. In all cases, with quantum yield changes the stopped-flow anisotropy signals cannot be fit with models consisting of exponentials. Case studies examining these effects are described for the protein folding/refolding transitions of Staphylococcal nuclease and phosphoglycerate kinase. A multiphasic DNA exonuclease reaction using bacteriophage T4 DNA polymerase is also examined. In all of these cases, combined analysis of both data types revealed insights into reaction mechanism, which could not be obtained by either data type in isolation. Quantum yields and steady-state anisotropies associated with transiently populated intermediate species can be resolved. The data analysis methodologies described allow characterization of multiphasic reactions in terms of internally consistent kinetic rates, quantum yields, and steady-state anisotropies.