A microchannel solution mixer for studying microsecond protein folding reactions

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 35-1000 mus time regime. The mixer is constructed by laser-machining 75-mum channels in a 127-mum-thick polyimide or polyetheretherketone polymer wafer. Mixing times of similar to25 to similar to50 mus can be achieved for a 1/10 dilution reaction of 8 M urea with flow rates of 10-20 mL/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. (C) 2005 American Institute of Physics.