The biological roles of heparin (HP) and heparan sulfate (HS) are mediated mainly through their interaction with proteins. In the present work, we provide a rapid method for screening HP/HS-protein interactions providing structural data on the key sulfo groups that participate in the binding. A library of polysaccharides structurally related to HP was prepared by immobilizing the biotinylated N-sulfated K5 polysaccharide (N-sulfoheparosan) on sensor chips followed by selective modification of this polysaccharide with enzymes that participate in HP/HS biosynthesis. The polysaccharides synthesized on the surface of the sensor chips differ in the number and position of sulfo groups present both on uronic acid and glucosamine residues. Surface plasmon resonance was used to measure the interaction of each member of this polysaccharide library with antithrombin III (ATIII), to afford structural information on sulfo groups required for this HP/HS-protein interaction. This method is viewed as widely applicable for the study of the structure-activity relationship (SAR) of HP/HS-protein interactions. (c) 2005 Elsevier Inc. All rights reserved.