Monitoring the unfolding and refolding of creatine kinase by capillary zone electrophoresis.
ABSTRACT Creatine kinase (ATP: creatine N-phosphotransferase; EC 188.8.131.52) plays a key role in the energy transport, muscle contraction, and reproduction of adenosine triphosphate (ATP). The activity of the enzyme is dependent on the correct folding of the peptide. We observed the unfolding and refolding processes of the creatine kinase of rabbit muscle, and concluded that traditional electrophoresis technology is unsuitable for the transient folding intermediates formed during protein unfolding and refolding. Capillary zone electrophoresis with diode array detection was used to monitor the unfolding and refolding of rabbit creatine kinase under different pretreatments and experimental conditions. This technique provides a simple, sensitive, and rapid approach to protein unfolding and refolding.
- SourceAvailable from: Vaclav Kasicka[Show abstract] [Hide abstract]
ABSTRACT: The article gives a comprehensive review on the recent developments in the applications of high-performance capillary electromigration methods, zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography, to analysis, preparation, and physicochemical characterization of peptides. The article presents new approaches to the theoretical description and experimental verification of electromigration behavior of peptides, covers the methodological aspects of capillary electroseparations of peptides, such as rational selection of separation conditions, sample preparation, suppression of peptide adsorption, new developments in individual separation modes, and new designs of detection systems. Several types of applications of capillary electromigration methods to peptide analysis are presented: conventional qualitative and quantitative analysis, purity control, determination in biomatrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid and sequence analysis, and peptide mapping of proteins. Some examples of micropreparative peptide separations are given and capabilities of capillary electromigration techniques to provide important physicochemical characteristics of peptides are demonstrated.Electrophoresis 02/2006; 27(1):142-75. · 3.16 Impact Factor