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Capillary electrophoresis frontal analysis for characterization of alpha(v)beta(3) integrin binding interactions

TitleCapillary electrophoresis frontal analysis for characterization of alpha(v)beta(3) integrin binding interactions
Publication TypeJournal Article
Year of Publication2008
AuthorsSun, Y, Cressman, S, Fang, N, Cullis, PR, Chen, DDY
JournalAnalytical Chemistry
Volume80
Pagination3105-3111
Date PublishedMay
Type of ArticleArticle
ISBN Number0003-2700
KeywordsANGIOGENESIS, BEHAVIOR, CONSTANTS, CYCLIC RGD PEPTIDES, DRUG-PROTEIN-BINDING, INTEGRIN ALPHA(V)BETA(3), PRINCIPLES, RECOGNITION, TRANSDUCTION
Abstract

The specific binding characteristics of alpha(v)beta(3) integrins with an arginine-glycine-aspartic-acid (RGD) containing fluorescently labeled cyclic peptide is investigated with capillary electrophoresis-frontal analysis method. The new algorithm used to calculate the binding constants and binding stoichiometry was derived without the assumptions made in the commonly used Scatchard Plot method, thus enabling the determination of specific binding parameters in the presence of nonspecific binding. The a,6,3 integrin, a membrane protein, was studied in solution, without the need of immobilization or any other kind of modification. An RGD containing fluorescently labeled cyclic pentapeptide is used as the ligand with both specific and nonspecific binding characteristics, and an arginine-alanine-aspartic-acid (RAD) containing peptide is used as the control for nonspecific binding. While a typical specific binding isotherm has a shape of a rectangular hyperbola, a nonspecific binding isotherm is linear in the same ligand concentration region. A 1:2 specific binding stoichiometry was revealed with the second binding having a similar affinity compared to the first binding event.

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