Dielectric resonator-based flow and stopped-flow EPR with rapid field scanning: A methodology for increasing kinetic information

We report methodology which combines recently developed dielectric resonato r-based, rapid-mix, stopped-flow EPR (appropriate for small aqueous, lossy samples) with rapid scanning of the external (Zeeman) magnetic held where t he 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 sp in-labeled species. We first used the ascorbyl radical as a test system whe re rapid scans triggered after flow was stopped provided "snapshots" of sim ultaneously evolving and interacting radical species, We monitored ascorbyl radical populations either as brought on by biologically damaging peroxyni trite oxidant or as chemically and kinetically interacting with a spectrosc opically overlapping nitroxide radical. In a different biophysical applicat ion, where a spin-label lineshape reflected rapidly changing molecular dyna mics of folding spin-labeled protein, rapid scan spectra were taken during how with different flow rates and correspondingly different times after the mixing-induced inception of protein folding. This flow/rapid scan method i s a means for monitoring early immobilization of the spin probe in the cour se of the folding process. (C) 1999 Academic Press.