Site-directed immobilization of glycoproteins on hydrazide-containing solid supports

Methods are described for the preparation and use of solid supports containing hydrazide functions for the immobilization of glycoproteins specifically through the oligosaccharide moieties. The solid supports are prepared from commercial "active ester" agarose by reaction with hydrazine hydrate. Glycoproteins are oxidized with sodium periodate, resulting in the production of aldehydes on the oligosaccharide moieties. Oxidized glycoprotein is then reacted with the hydrazide-derivatized solid support to produce stable hydrazone linkages. Data are presented for the optimization of binding of oxidized glycoprotein to hydrazide-derivatized agarose. Agarose hydrazide/glycoprotein gels were shown to be stable from pH 3 to 10 and activity studies using immobilized avidin show that this method of immobilization results in an increased "specific activity" of bound protein when compared with standard methods of immobilization.