Avidin is a heparin-binding protein. Affinity, specificity and structural analysis.
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.
Kett
Warren C
WC
Molecular Immunology Laboratory, School of Biomedical Sciences, Curtin University of Technology, Level 5 MRF Building, Rear 50 Murray Street, Perth, Western Australia 6000, Australia.
Osmond
Ronald I W
RI
Moe
Lily
L
Skett
Spencer E
SE
Kinnear
Beverley F
BF
Coombe
Deirdre R
DR
D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001360:Avidin / Q000031:analogs & derivatives* / Q000737:chemistry*; D001798:Blood Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D006025:Glycosaminoglycans / Q000737:chemistry; D006493:Heparin / Q000032:analysis / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014407:Tumor Cells, Cultured
D023181:Antimicrobial Cationic Peptides; D001360:Avidin; D001798:Blood Proteins; D002352:Carrier Proteins; D016650:Fluorescein-5-isothiocyanate; D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; D012710:Serum Albumin, Bovine; C062991:AZU1 protein, human; C036963:fluorescein isothiocyante avidin