title: Monitoring equilibria and kinetics of protein folding/unfolding reactions by capillary zone electrophoresis.
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 x 10(-4) cm(2) 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.
authors.affiliation: Department of Agricultural and Industrial Biotechnologies, University of Verona, Strada le Grazie No. 15, Cà Vignal, Verona, 37134, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Verzola
authors.forename: F
authors.initials: F
authors.lastname: Chiti
authors.forename: G
authors.initials: G
authors.lastname: Manao
authors.forename: P G
authors.initials: PG
authors.lastname: Righetti
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry*; D002021:Buffers; D004151:Dipeptides; D019075:Electrophoresis, Capillary / Q000379:methods*; D007526:Isoelectric Point; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014508:Urea; D000097024:Acylphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D002021:Buffers; D004151:Dipeptides; D014508:Urea; D000097024:Acylphosphatase