ABSTRACT
The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.
Subject(s)
Avidin/analogs & derivatives , Avidin/chemistry , Blood Proteins/chemistry , Carrier Proteins/chemistry , Fluorescein-5-isothiocyanate/analogs & derivatives , Heparin/chemistry , Animals , Antimicrobial Cationic Peptides , Cell Membrane/chemistry , Cell Membrane/metabolism , Flow Cytometry , Glycosaminoglycans/chemistry , Heparin/analysis , Heparitin Sulfate/chemistry , Hydrogen-Ion Concentration , Protein Binding , Serum Albumin, Bovine/chemistry , Tumor Cells, CulturedABSTRACT
Surface plasmon resonance (SPR) biosensors such as the BIAcore 2000 are a useful tool for the analysis of protein-heparin interactions. Generally, biotinylated heparin is captured on a streptavidin-coated surface to create heparinized surfaces for subsequent binding analyses. In this study we investigated three commonly used techniques for the biotinylation of heparin, namely coupling through either carboxylate groups or unsubstituted amines along the heparin chain, or through the reducing terminus of the heparin chain. Biotinylated heparin derivatives were immobilized on streptavidin sensor chips and several heparin-binding proteins were examined. Of the surfaces investigated, heparin attached through the reducing terminus had the highest binding capacity, and in some cases had a higher affinity for the proteins tested. Heparin immobilized via intrachain bare amines had intermediate binding capacity and affinity, and heparin immobilized through the carboxylate groups of uronic acids had the lowest capacity for the proteins tested. These results suggest that immobilizing heparin to a surface via intrachain modifications of the heparin molecule can affect the binding of particular heparin-binding proteins.
