A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins.
A surface plasmon resonance-based solution affinity assay is described for measuring the K(d) of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin-streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar K(d) values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications.
Cochran
Siska
S
Drug Design Group, Progen Pharmaceuticals Ltd, Darra, QLD, Australia.
Li
Cai Ping
CP
Ferro
Vito
V
D002352:Carrier Proteins / Q000378:metabolism*; D054412:Chemokine CCL8 / Q000378:metabolism; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D002352:Carrier Proteins; D054412:Chemokine CCL8; D016220:Fibroblast Growth Factor 1; D016222:Fibroblast Growth Factor 2; D006497:Heparan Sulfate; D016209:Interleukin-8; D042461:Vascular Endothelial Growth Factor A