A microchannel solution mixer for studying microsecond protein folding reactions

Abstract

Many proteins fold through intermediates that are populated in the submillisecond time regime. To monitor directly the formation of these kinetic intermediates, we have developed a simple, robust, easy to assemble continuous flow mixer for studying folding reactions in the 35–1000μs time regime. The mixer is constructed by laser-machining 75-μm channels in a 127-μm-thick polyimide or polyetheretherketone polymer wafer. Mixing times of ∼25to∼50μs can be achieved for a 1∕10 dilution reaction of 8M urea with flow rates of 10–20mL∕min. CCD-based steady-state and time-correlated single-photon-counting-based fluorescence detection strategies are described. Preliminary results on the early events in the refolding of cytochrome c are presented.