A highly stable covalent conjugated heparin biochip for heparin-protein interaction studies.

Heparin is a proteoglycan composed of highly sulfated linear polysaccharides of alternating uronic acid and glucosamine that interacts with a wide variety of proteins and peptides (1). Heparin and the structurally related heparan sulfate are the most acidic polysaccarides in the human body and, as a result, interact with many cationic proteins, giving rise to myriad biological activities (2). Some of these interactions have received extensive attention in recent years, including heparin’s binding to growth factors (3, 4) influencing angiogenesis and other proliferation-dependent processes, and its binding to the ectodomain proteins of pathogens influencing infection (5). Surface plasmon resonance (SPR) spectroscopy has become one of several established methods for measuring biomolecular interactions (6). SPR measures binding interactions on the surface of a biosensor chip, and SPR biosensors have been successfully used for quantitative modeling of heparin–protein interactions (3, 7–9). These experiments require the immobilization of either heparin or the heparin-binding protein on the surface of a biosensor chip, over which its binding partner (heparin-binding protein or heparin) is passed. While a number of immobilization chemistries have been developed to immobilize proteins for SPR (6), this may be problematic as proteins differ with respect to ease of covalent attachment to a surface with retention of native conformation, accessibility of active sites, and bioactivity. In natural biological systems, heparan sulfate is found immobilized on the cell surface through its core protein (10), and captures heparin-binding proteins that flow over the cell surface. Modeling this interaction by SPR would best be achieved by immobilizing heparin/heparan sulfate rather than the heparin-binding protein. However, the presence of only a single reducing-end amine group in glycosaminoglycans such as heparin and heparan sulfate makes the usual amine chemistry-based immobilization chemistries problematic, and we have been exploring alternative approaches. In all previous heparin–protein interaction studies using SPR, heparin was immobilized through a bridging avidin–biotin system (3, 7, 8), biotinylated heparin being bound to avidin or streptavidin immobilized on the chip surface. However, studies in our laboratory demonstrated that many heparin-binding proteins interact nonspecifically with avidin and streptavidin, making the use of such heparin-containing biochips problematic (9). Furthermore, we also found that the streptavidin–biotin heparin chip was not stable, especially when the heparin-binding protein is tightly bound and harsh regeneration conditions (e.g., washing the surface with NaOH) are required, leading to damage to the linkage between the ligand and surface. Our laboratory (9) and others (11) have previously immobilized albumin–heparin conjugates on functionalized polystyrene surfaces to study heparin–protein interactions. In the present study, we describe a new method for the covalent immobilization of heparin on a biochip using a preformed albumin–heparin conjugate and its utility for studying interactions with heparin– binding proteins.