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

A method is described here for studying conformational transitions of proteins due to denaturing agents: capillary zone electrophoresis (CZE) in acidic, isoelectric buffers. The sample is run in 50 mM isoelectric glutamic acid (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 of 2.63 × 10−4 cm2 V−1 s−1. By studying the unfolding kinetics, as a function of time of incubation in 7 M urea, it was possible to measure the rate constant of the unfolding reaction, estimated to be 0.00030 ± 0.00006 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 unfolding 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 urea as midpoint of the unfolding curve.