Modification and introduction of various radioactive labels into the sialic acid moiety of sialoglycoconjugates

A method for modifying and isotopic labeling the sialyl moiety of sialoglycoproteins is described. The basis of the procedure is the reductive amination of the exocyclic aldehyde group, generated on sialic acid by mild periodate oxidation, with a variety of amino compounds and sodium cyanoborohydride. Optimal conditions were selected to obtain maximum modification of sialic acid and minimal non-specific incorporation of the amino compound (glycine). The glycine modified model glycoproteins (α₁-acid glycoprotein, fetuin) yielded single homogenous peaks upon gel filtration and on ion exchange chromatography. On gel electrophoresis a major band accounting for 92–98% of the modified glycoprotein and two minor bands consisting of dimers and trimers of the glycoprotein were observed. The modification did not alter the ability of the sialoglycoproteins to bind to wheat germ agglutinin-Sepharose or to interact with antibodies. The modified sialic acid was only partially released by mild acid hydrolysis suggesting that the introduction of an amino compound into the polyol chain of sialic acid has a stabilizing effect on the ketosidic linkage of the sugar. Interestingly, the modification rendered the sialic acid resistant to a variety of sialidases. The potential uses of this modification procedure include 1) the introduction of different isotopic labels (³H,¹⁴C,³⁵S,¹²⁵I) into the sialic acid moiety of glycoproteins; 2) the preparations of biologically active sialoglycoprotein (hormones, enzymes, co-factors) with increased circulating half-lives in animals; 3) preparation of substrates to search for endoglycosidases; 4) the direct comparison of sialoglycoprotein patterns obtained in small amounts from normal and pathological cells or tissues, and 5) the isolation and purification of cell surface sialoglycoproteins.