Mobility Moment Analysis of Molecular Interactions by Capillary Electrophoresis
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- 作者：Kiyohito Shimura ; Noboru Uchiyama ; Manabu Enomoto ; Hiroyuki Matsumoto ; and Ken-ichi Kasai
- 刊名：Analytical Chemistry
- 出版年：2005
- 出版时间：January 15, 2005
- 年：2005
- 卷：77
- 期：2
- 页码：564 - 572
- 全文大小：130K
- 年卷期：v.77,no.2(January 15, 2005)
- ISSN：1520-6882
文摘
A new method for the quantitative evaluation of molecularinteractions that are observed in electrophoresis is described. One component taking part in the interaction islabeled with a fluorescent dye and is subjected to capillaryzone electrophoresis with fluorescence detection in thepresence or absence of an unlabeled interacting component. Fluorescence signals are collected at constant timeintervals, and the electropherograms are converted torepresent the fluorescence signal against mobility. Afterbaseline subtraction, the first statistical moment of fluorescence signals on the mobility axis is calculated. Thismoment represents the average mobility of a labeledcomponent. The change in the mobility moment in thepresence and absence of the unlabeled component is usedto evaluate the degree of saturation of the binding site ofa labeled molecule with an unlabeled molecule. Mixturesof fluorescence-labeled protein (Fab' fragment of antibodyor concanavalin A) and its unlabeled interacting partner(
1-antitrypsin or succinylated ovalbumin, respectively)at various concentrations were injected into a bare-silicacapillary, and zone electrophoresis was carried out. Thechange in the mobility moment of the fluorescence-labeledmolecules was used to determine the dissociation constants of the complexes. The determined constants arecomparable to those obtained by a well-established method,that is, an analysis based on the peak height of thecomplex. Since the mobility moment analysis is notaffected by the total intensity of the signals, it should beadvantageous in analyses in which multiple capillaries areused, in which the injection volume and the sensitivity ofdetection might be more difficult to control at constantvalues. The mobility moment analysis also has advantagesfor the analysis of heterogeneous samples, since theidentification of peaks is not necessarily required.
1-antitrypsin or succinylated ovalbumin, respectively)at various concentrations were injected into a bare-silicacapillary, and zone electrophoresis was carried out. Thechange in the mobility moment of the fluorescence-labeledmolecules was used to determine the dissociation constants of the complexes. The determined constants arecomparable to those obtained by a well-established method,that is, an analysis based on the peak height of thecomplex. Since the mobility moment analysis is notaffected by the total intensity of the signals, it should beadvantageous in analyses in which multiple capillaries areused, in which the injection volume and the sensitivity ofdetection might be more difficult to control at constantvalues. The mobility moment analysis also has advantagesfor the analysis of heterogeneous samples, since theidentification of peaks is not necessarily required.