Preparation of High Molecular Weight Glycopeptides and Oligosaccharides (Erythroglycan) from Human Erythrocytes

This chapter presents procedure for preparation of high molecular weight glycopeptides and oligosaccharides (erythroglycan) from human erythrocytes. Human erythrocyte membranes are prepared and delipidated by chloroform-methanol and water-n-butanol extraction. Oligosaccharides are released from this dry, lipid-free residue by hydrazinolysis, or the corresponding glycopeptides are prepared by exhaustive digestion with Pronase and cleavage of O-glycosidic bonds by mild alkaline borohydride treatment. The high molecular weight oligosaccharides or glycopeptides are separated from the smaller glycans by gel filtration giving a molecular weight profile of the total glycopeptides of the erythrocyte. The pooled fraction of glycopeptides of molecular weight 7000 to 12,000 constitutes the erythroglycan fraction. In an optional modification of this method, the cells or membranes are pretreated with trypsin, which removes most of the lower molecular weight protein-bound saccharide chains but none of the erythroglycan. In this modification the low molecular weight glycopeptides are removed prior to further processing by washing the trypsinized cells.