Study of hyaluronan-binding proteins and receptors using iodinated hyaluronan derivatives

This chapter detailed methodology for the purification of high molecular weight HA, as well as procedures to fragment the HA to prepare large oligosaccharides in the range of 40,000-80,000 Da. The aforementioned procedures used to prepare HA-alkylamine and HA-Bolton-Hunter adducts, as well as ¹²⁵I-labeled HA, have been very reproducible, and the latter preparations are of adequate length to retain high-affinity interactions and specific binding, e.g., to human fibrinogen and HARE. For example, we were able to isolate, characterize, and clone the rat HARE using ¹²⁵I-labeled HA initially with the dot blot assay to monitor solubilization and partial purification, and later with the ligand blot assay, to identify the protein after SDS-PAGE. The ligand blot assay enabled us to determine that HARE is actually present as two discrete isoreceptors of different molecular masses. These techniques should provide a means to analyze purification strategies and to characterize additional HA receptors and binding proteins involved in a variety of physiologic processes.