On the adsorption of hyaluronan and ICAM-1 to modified hydrophobic resins

Hyaluronan is a negatively charged glycosaminoglycan that occurs in connective tissue and has a wide range of mechanical and cell biological functions. The purpose of this study was to utilize affinity chromatography resins for purification of detergent (Triton X-100) solubilized hyaluronan binding proteins from liver, the major organ of hyaluronan clearance from the blood. However, during these studies we made the unexpected finding that hyaluronan binds to Sepharose substituted with a hexamethylene chain, a commonly used spacer arm in affinity chromatography resins, capped with either a terminal primary amine or a terminal acetoamido group. Hyaluronan did not bind the hydrophobic resins hexyl- or octyl-Sepharose under the same conditions. It was also found that rat liver intercellular adhesion molecule-1 binds to resins containing the hexamethylene spacer arm, an interaction which could be inhibited with free hyaluronan oligosaccharides. Finally, we have determined that resins with ethylene spacer arms show no affinity for hyaluronan and can therefore be used to immobilize hyaluronan for chromatography of hyaluronan binding proteins. By using this resin we have purified two proteins of approximately 200 and 400 kDa from rat liver endothelial cells. In summary, this study demonstrates the efficacy of certain "capped-hydrophobic" resins for binding hyaluronan; these resins may provide a novel means for the study and/or purification of this glycosaminoglycan. This study further demonstrates the importance of the careful design of appropriate affinity columns for the specific purification of hyaluronan binding proteins.