Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization.
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.
Osmond
Ronald I W
RI
Molecular Immunology Laboratory, School of Biomedical Sciences, Curtin University of Technology, Level 5 MRF Building, Rear 50 Murray Street, Perth, WA 6000, Australia.
Kett
Warren C
WC
Skett
Spencer E
SE
Coombe
Deirdre R
DR
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D001360:Avidin / Q000378:metabolism*; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods; D001710:Biotin / Q000737:chemistry; D005944:Glucosamine / Q000737:chemistry; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007781:Lactoferrin / Q000378:metabolism*; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000295:instrumentation* / Q000379:methods; D013499:Surface Properties; D013552:Swine; D013917:Thrombin / Q000378:metabolism*; D014574:Uronic Acids / Q000737:chemistry
D014574:Uronic Acids; D001360:Avidin; D001710:Biotin; D000990:Antithrombin III; D006493:Heparin; D019809:Streptavidin; D007781:Lactoferrin; D013917:Thrombin; D005944:Glucosamine