Monitoring the unfolding and refolding of creatine kinase by capillary zone electrophoresis
School of Life Science and Engineering, Tsinghua University, Beijing, People's Republic of China.Journal of capillary electrophoresis and microchip technology 01/2003; 8(1-2):7-10.
Creatine kinase (ATP: creatine N-phosphotransferase; EC 184.108.40.206) 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.
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ABSTRACT: Protein denaturation and aggregation are well-known problems in the pharmaceutical industry. As the protein aggregates, it loses its biological activity and creates problems in its administration to patients. In this paper, we explore the use of aqueous two-phase systems, capillary zone electrophoresis, and dynamic light scattering for the monitoring of protein denaturation and aggregation. Our studies focus on human IgG and HSA. Capillary zone electrophoresis was used to monitor changes in the charge to size ratio of the proteins upon denaturation and dynamic light scattering was used to detect the presence of any aggregates and to monitor the size of the proteins. The information obtained from aqueous two-phase partitioning is similar to that obtained from capillary zone electrophoresis. The simplicity of aqueous two-phase system and its low cost (compared to the other analytical techniques) suggest that it can be routinely used for the quality control of some pharmaceutical preparations.
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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.
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