Analytical Sciences
Online ISSN : 1348-2246
Print ISSN : 0910-6340
ISSN-L : 0910-6340
Nano-Scale Monitoring of the Thermally-Induced Unfolding of Proteins Using Capillary Electrophoresis with In-Column Incubation
Yasushi ISHIHAMAYoshiya ODANaoki ASAKAWAMasahiro IWAKURA
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1997 Volume 13 Issue 6 Pages 931-938

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Abstract

Based on the difference between the electrophoretic mobility of the native protein and that of the unfolded one, the thermally-induced unfolding transition of proteins was analyzed by capillary electrophoresis (CE) using an in-column incubation method. In this method, the required amount of sample could be reduced to 100ng because the unfolding process occurred not in the sample vial, but in the separation capillary after the sample injection. The thermodynamic parameters of the unfolding transition process were obtained by analyzing the dependence of the electrophoretic mobilities on the temperature inside the capillary controlled by the thermostated water. The temperature inside the capillary was measured by an easy and rapid method on the basis of the dependence of the electrical current on the applied voltage. The electrophoretic mobility was converted to a temperature-independent parameter using the average of the electrical current through the analysis. In this work, the unfolding processes of several proteins were investigated. Although the apparent transition temperatures by CE were generally consistent with those obtained by far-UV CD using a two-state transition model, these two methods had different detection selectivity for the change in the protein structures. The CE method would offer additional information for the unfolding processes of proteins.

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© The Japan Society for Analytical Chemistry
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