Variable velocity liquid flow EPR applied to submillisecond protein folding

We have developed a variable velocity, rapid-mix, continuous-flow method fo r observing and delineating kinetics by dielectric resonator-based electron paramagnetic resonance (EPR). The technology opens a new facet for kinetic study of radicals in liquid at submillisecond time resolution. The EPR sys tem (after Sienkiewicz, A., K. Qu, and C. P. Scholes. 1994. Rev. Sci. Instr um. 65:68-74) accommodated a miniature quartz capillary mixer with an appro ximate to 0.5 mu L delivery volume to the midpoint of the EPR-active zone. The flow velocity was varied in a preprogrammed manner, giving a minimum de livery time of approximate to 150 mu s. The mixing was efficient, and we co nstructed kinetics in the 0.15-2.1-ms time range by plotting the continuous wave EPR signal taken during flow versus the reciprocal of flow velocity. We followed the refolding kinetics of iso-1-cytochrome c spin-labeled at Cy steine 102. At 20 degrees C, upon dilution of guanidinium hydrochloride den aturant, a fast phase of refolding was resolved with an exponential time co nstant of 0.12 ms, which was consistent with the "burst" phase observed by optically detected flow techniques. At 7 degrees C the kinetic refolding ti me of this phase increased to 0.5 ms.