Dielectric resonator-based flow and stopped-flow EPR with rapid field scanning: A methodology for increasing kinetic information.
Dielectric resonator-based flow and stopped-flow EPR with rapid field scanning: A methodology for increasing kinetic information.
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DOI：
10.1006/jmre.1998.1630
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发表时间：
1999-02
影响因子：
2.2
通讯作者：
A. Sienkiewicz;A. M. da Costa Ferreira;B. Danner;C. Scholes
中科院分区：
文献类型：
--
作者：
A. Sienkiewicz;A. M. da Costa Ferreira;B. Danner;C. Scholes
我们报告的方法，它结合了最近开发的介电谐振器为基础的，快速混合，停止流动EPR（适用于小，水性，有损耗的样品）与快速扫描的外部（塞曼）磁场的扫描是预编程发生在选定的时间后，开始流动。这种方法提供了光谱信息的补充，通过停流EPR在单场获得的，并与低反应物的使用，它产生了更多的图形洞察自由基和自旋标记的物种的时间演变。我们首先使用抗坏血酸自由基作为测试系统，其中在流动停止后触发的快速扫描提供了同时进化和相互作用的自由基物种的“快照”。我们监测抗坏血酸自由基的人口无论是生物破坏过氧亚硝酸盐氧化剂或化学和动力学相互作用的光谱重叠的氮氧自由基。在不同的生物物理应用中，自旋标记的线形反映了快速变化的分子动力学折叠自旋标记的蛋白质，快速扫描光谱在流动过程中采取不同的流速和相应的不同时间后，混合诱导的蛋白质折叠的开始。该流动/快速扫描方法是用于在折叠过程中监测自旋探针的早期固定的手段。
We report methodology which combines recently developed dielectric resonator-based, rapid-mix, stopped-flow EPR (appropriate for small, aqueous, lossy samples) with rapid scanning of the external (Zeeman) magnetic field where the scanning is preprogrammed to occur at selected times after the start of flow. This methodology gave spectroscopic information complementary to that obtained by stopped-flow EPR at single fields, and with low reactant usage, it yielded more graphic insight into the time evolution of radical and spin-labeled species. We first used the ascorbyl radical as a test system where rapid scans triggered after flow was stopped provided "snapshots" of simultaneously evolving and interacting radical species. We monitored ascorbyl radical populations either as brought on by biologically damaging peroxynitrite oxidant or as chemically and kinetically interacting with a spectroscopically overlapping nitroxide radical. In a different biophysical application, where a spin-label lineshape reflected rapidly changing molecular dynamics of folding spin-labeled protein, rapid scan spectra were taken during flow with different flow rates and correspondingly different times after the mixing-induced inception of protein folding. This flow/rapid scan method is a means for monitoring early immobilization of the spin probe in the course of the folding process.