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.
Monitoring equilibria and kinetics of proteins folding/unfolding reactions by capillary zone electrophoresis / B. Verzola; F. Chiti; G. Manao; ; P. Righetti. - In: ANALYTICAL BIOCHEMISTRY. - ISSN 0003-2697. - STAMPA. - 282:(2000), pp. 239-244. [doi:10.1006/abio.2000.4620]
Monitoring equilibria and kinetics of proteins folding/unfolding reactions by capillary zone electrophoresis
CHITI, FABRIZIO;MANAO, GIAMPAOLO;
2000
Abstract
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.I documenti in FLORE sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.