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.
Bibliographical noteFunding Information:
The research described here has been supported by National Institutes of Health Grant GM35978 from the National Institute of General Medical Sciences.