Nano-Scale Monitoring of the Thermally-Induced Unfolding of Proteins Using Capillary Electrophoresis with In-Column Incubation

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 100 ng 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.

Stability of proteins in the presence of carbohydrates; experiments and modeling using scaled particle theory

“…CE offers the advantages of high sensitivity, minute sample size, quick analysis time, and high resolution [37]. Thermodynamic parameters determined from CE have been shown to be in good quantitative agreement with those determined from CD and calorimetry [30,31]. In this study the effects of sucrose and fructose on the thermodynamics of unfolding of RNase A and α-LA were measured by monitoring the electrophoretic mobility (ν elec ) of the two proteins as a function of temperature.…”

“…Recently a growing number of studies have utilized CE to monitor the folding/unfolding transitions of proteins [20,[29][30][31][32][33][34][35][36]. CE offers the advantages of high sensitivity, minute sample size, quick analysis time, and high resolution [37].…”

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“…For proteins unfolding in the fast time regime, these two opposing effects can generate unfolding transitions that are difficult to interpret, unless independent corrections for apparent viscosity changes are implemented. For example, Ishihama et al [48] used the mobility to current ratio in CE thermal-induced protein unfolding studies to normalize for viscosity changes in order to derive more accurate thermodynamic parameters.…”

“…Ishihama et al performed thermalinduced unfolding on cyt. c and a series of other proteins with subsequent validation by circular dichroism [48]. This was the first report of dynamic unfolding by CE, referred to as direct in-capillary incubation during electromigration, which highlighted the unique benefit of studying protein unfolding in the fast-time regime.…”

“…Multiplexed information regarding protein conformational changes can be gained by the integration of these detectors to CE methods. Albumin Thermal unfolding Conformational study [48] [ 64,65] Alcohol dehydrogenase…”