Variable Velocity Liquid Flow EPR Applied to Submillisecond Protein Folding

We have developed a variable velocity, rapid-mix, continuous-flow method for 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 system (after Sienkiewicz, A., K. Qu, and C. P. Scholes. 1994. Rev. Sci. Instrum. 65:68–74) accommodated a miniature quartz capillary mixer with an ≈0.5 μL delivery volume to the midpoint of the EPR-active zone. The flow velocity was varied in a preprogrammed manner, giving a minimum delivery time of ≈150 μs. The mixing was efficient, and we constructed 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 Cysteine 102. At 20°C, upon dilution of guanidinium hydrochloride denaturant, a fast phase of refolding was resolved with an exponential time constant of 0.12 ms, which was consistent with the “burst” phase observed by optically detected flow techniques. At 7°C the kinetic refolding time of this phase increased to 0.5 ms.