Monitoring equilibria and kinetics of protein folding/unfolding reactions by capillary zone electrophoresis

A method is described here for studying conformational transitions of prote ins due to denaturing agents: capillary zone electrophoresis (CZE) in acidi c, isoelectric buffers. The sample is run in 50 mM isoelectric glutamic aci d (pH = pI = 3.2) added with 1 mM oligoamine (tetraethylene pentamine) for quenching protein interaction to the capillary wall (final pH 3.3). Muscle acylphosphatase (AcP), in this buffer, exhibited a free solution mobility o f 2.63 x 10(-4) cm(2) V-1 s(-1). By studying the unfolding kinetics, as a f unction of time of incubation in 7 IM urea, it was possible to measure the rate constant of the unfolding reaction, estimated to be 0.00030 +/- 0.0000 6 s(-1). The same measurements, when repeated via spectroscopic monitoring of intrinsic fluorescence, gave a value of 0.00034 +/- 0.00002 s(-1), thus in excellent agreement with CZE data By equilibrium unfolding CZE studies, it was possible to construct the typical sigmoidal transition of unfoIding vs urea molarity: the midpoint of this transition, at which the folded and unfolded states should be equally populated, was estimated to be at 4.56 M urea. Similar experiments by fluorometric analysis gave a value of 4.60 M u rea as midpoint of the unfolding curve. (C) 2000 Academic Press.