Capillary electrophoresis frontal analysis for characterization of alphavbeta3 integrin binding interactions.
The specific binding characteristics of alphavbeta3 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 alphavbeta3 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.
Sun
Ying
Y
Department of Chemistry, Centre for Drug Research and Development, University of British Columbia Vancouver, BC, Canada V6T 1Z3.
Cressman
Sonya
S
Fang
Ning
N
Cullis
Pieter R
PR
Chen
David D Y
DD
D000465:Algorithms; D003198:Computer Simulation; D003627:Data Interpretation, Statistical; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D039302:Integrin alphaVbeta3 / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry*; D011485:Protein Binding
D005456:Fluorescent Dyes; D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D010456:Peptides, Cyclic; C047981:arginyl-glycyl-aspartic acid