Abstract
We have developed a hydrodynamic fast mixer capable of following protein folding kinetics. Our design, which comprises two co-centered fused silica capillaries, employs three dimensional focusing of the sample channel to create a laminar flow. Construction of the mixer is simple and inexpensive, requiring no specialized equipment or techniques. In characterizing this new mixer, we have also developed a novel sample flow rate calibration method utilizing the fluorescence decay of excited europium (Eu) beads. To demonstrate the mixer's application in biophysical studies, we have used this mixer to study apomyoglobin (apoMb) folding kinetics by inducing folding or unfolding via a rapid change in the pH of the protein's environment. We probed the protein with 1-anilino-8-naphthalene sulfonate (1,8-ANS), and observed the fluorescence using fluorescence confocal microscopy. Scans along the z-axis of the sample channel were collected and analyzed to identify the location of folding or unfolding events and timescale over which these events occurred. Our ultimate goal is to develop a kinetic model of apoMb folding. If the model agrees with previous work on apoMb folding kinetics, we may then use our mixer to characterize the folding and reaction kinetics of other protein systems.
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