Directional Immobilization of Heparin Onto Beaded Supports

Heparin was immobilized in a defined orientation on Sepharose, agarose, and polyacrylamide supports by coupling through its reducing end. This is expected to mimic the attachment of heparin to the protein core in the naturally occurring proteoglycan and impart better ligand binding efficiency by exposing all the binding sites available in the naturally occurring heparin. The coupling chemistry was accomplished by modifying heparin at its reducing end to introduce reactive functionality that can react with appropriately functionalized supports. Three reducing end modified heparins were synthesized and characterized: 2,6-diaminopyridinyl heparin, containing a reactive amino group at the reducing end; ω-hydrazido-adipyl-azo heparin, containing a hydrazido group at the reducing end; and heparin lactone, containing a reactive ester functionality at the reducing end. These heparin derivatives were then reacted with the supports to give directionally immobilized heparin using different coupling chemistries: coupling of reducing end modified heparins to amine-containing supports (i.e., ω-aminohexyl Sepharose and omega-aminobutyl agarose), hydrazide-containing supports (i.e., Emphaze hydrazide), and activated carboxy-containing supports (i.e., activated 6-aminohexanoic acid Sepharose, Emphaze azlactone). The heparinized matrices were prepared and analyzed for their heparin content and protamine binding capacity.