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 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.
Sienkiewicz
A
A
Institute of Physics, Al. Lotnikow 32, Warsaw, 02-668, Poland.
da Costa Ferreira
A M
AM
Danner
B
B
Scholes
C P
CP
D000975:Antioxidants; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D003683:Dehydroascorbic Acid / Q000031:analogs & derivatives; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods; D005609:Free Radicals / Q000032:analysis* / Q000737:chemistry*; D008280:Magnetics; D009566:Nitrates; D009589:Nitrogen Oxides; D016877:Oxidants; D017510:Protein Folding; D011673:Pulsatile Flow; D029701:Saccharomyces cerevisiae Proteins; D013113:Spin Labels