Interaction of soluble and surface-bound heparin binding growth-associated molecule with heparin.
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 deltaH of -30 kcal/mole corresponding to a dissociation constant (Kd) 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 Kd 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.
Fath
M
M
Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City 52242, USA.
VanderNoot
V
V
Kilpeläinen
I
I
Kinnunen
T
T
Rauvala
H
H
Linhardt
R J
RJ
D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D016207:Cytokines / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006493:Heparin / Q000378:metabolism*; D007700:Kinetics; D013552:Swine; D013816:Thermodynamics; D013997:Time Factors
D002352:Carrier Proteins; D016207:Cytokines; C066845:pleiotrophin; D006493:Heparin