| An understanding of how complex carbohydrates exhibit biological activities by interacting with proteins is of great physiologic importance. Protein-carbohydrate interactions underlie many aspects of cellular recognition including cell adhesion, trafficking, apoptosis, and the immune response. A better understanding of the structural and kinetic basis for the ability of heparin to interact with proteins of biological importance should facilitate the design of heparin analogs having potential therapeutic value.;The current study seeks to investigate the affinity & kinetic features of GAG-protein interactions. The major goal is to examine the interactions of heparin with a variety of biologically important proteins, including cytokines, chemokines, and complement proteins. Surface plasmon resonance (SPR) is used to study these interactions. NMR spectroscopy, affinity chromatography, and gel electrophoresis were used in the ligand preparation.;A secondary goal of this research is to develop a reliable SPR biosensor chip surface, on which commercial heparin & heparan sulfate as well as GAGS isolated from animal organs could be immobilized. This new chip surface should reduce or eliminate non-specific interaction. The knowledge obtained from these experiments will be exploited to better analyze the carbohydrate-protein interactions in future experiments.;These studies demonstrate that heparin interacts with the complement proteins C1INH, C1-9, factor I, factor H, factor B and factor P. Cytokines and chemokines including interleukin (IL)-6, IL-10, IFN-gamma and TNF-alpha were also shown by SPR to interact with heparin. The studies are the first to demonstrate the quantitative information in the form of affinity constants and on-rates and off-rates for the formation of these complexes using SPR biosensors in place of conventional solid-phase assays. A major advantage of biosensors for examining macromolecular interaction is that the formation and breakdown of complexes are monitored in real time, offering the possibility to determine the interaction mechanism and kinetic rate constants associated with a binding event. This information is essential for understanding how heparin and structurally related GAGs regulate these biological processes involving these proteins. Furthermore, this study provides insight required for the rational design of new therapeutic agents capable of regulating the functions of these proteins. |
