Osmond Ronald I W, Kett Warren C, Skett Spencer E, Coombe Deirdre R
Molecular Immunology Laboratory, School of Biomedical Sciences, Curtin University of Technology, Level 5 MRF Building, Rear 50 Murray Street, Perth, WA 6000, Australia.
Anal Biochem. 2002 Nov 15;310(2):199-207. doi: 10.1016/s0003-2697(02)00396-2.
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.
诸如BIAcore 2000之类的表面等离子体共振（SPR）生物传感器是分析蛋白质-肝素相互作用的有用工具。一般来说，生物素化的肝素被捕获在链霉亲和素包被的表面上，以创建肝素化表面用于后续的结合分析。在本研究中，我们研究了三种常用的肝素生物素化技术，即通过肝素链上的羧基或未取代的胺进行偶联，或通过肝素链的还原末端进行偶联。将生物素化的肝素衍生物固定在链霉亲和素传感器芯片上，并检测了几种肝素结合蛋白。在所研究的表面中，通过还原末端连接的肝素具有最高的结合能力，并且在某些情况下对测试的蛋白质具有更高的亲和力。通过链内裸胺固定的肝素具有中等的结合能力和亲和力，而通过糖醛酸的羧基固定的肝素对测试的蛋白质的结合能力最低。这些结果表明，通过肝素分子的链内修饰将肝素固定到表面上会影响特定肝素结合蛋白的结合。
