Document Detail


Monitoring equilibria and kinetics of protein folding/unfolding reactions by capillary zone electrophoresis.
MedLine Citation:
PMID:  10873279     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
B Verzola; F Chiti; G Manao; P G Righetti
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical biochemistry     Volume:  282     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2000 Jul 
Date Detail:
Created Date:  2000-08-21     Completed Date:  2000-08-21     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  239-44     Citation Subset:  IM    
Copyright Information:
Copyright 2000 Academic Press.
Affiliation:
Department of Agricultural and Industrial Biotechnologies, University of Verona, Strada le Grazie No. 15, Cà Vignal, Verona, 37134, Italy.
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MeSH Terms
Descriptor/Qualifier:
Acid Anhydride Hydrolases / chemistry*
Buffers
Dipeptides
Electrophoresis, Capillary / methods*
Isoelectric Point
Kinetics
Muscles / enzymology
Protein Folding*
Spectrometry, Fluorescence
Urea
Chemical
Reg. No./Substance:
0/Buffers; 0/Dipeptides; 57-13-6/Urea; EC 3.6.-/Acid Anhydride Hydrolases; EC 3.6.1.7/acylphosphatase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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