The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a ΔH of -30 kcal/mole corresponding to a dissociation constant (K(d)) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12-16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a K(d) of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of >14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction.