Influence of type of linkage and spacer on the interaction of beta-galactoside-binding proteins with immobilized affinity ligands

Journal/Book: Anal-Biochem 189 (1), 91-94. 1990;

Abstract: Affinity chromatography provides a powerful tool for isolation ofcarbohydrate-binding proteins. However, the choice of the ligand andspacer has an important impact on effectiveness. The influence ofseveral different ligands on qualitative and quantitative aspects of thepurification of two beta-galactoside-specific lectins has beenevaluated. Sepharose was modified by coupling four types ofneoglycoproteins (galactosylated or lactosylated bovine serum albuminwith increasing sugar content) and two naturally occurringasialoglycoproteins at similar densities. Carbohydrate ligands atessentially equal density were made accessible to the lectins by sevencommonly used methods. The yield of mistletoe lectin was high whenlactosylated neoglycoproteins were used for separation. For these resinsthe sugar incorporation exceeded 10 sugar groups per protein carriermolecule. The yield was similarly high with the asialoglycoproteins andwith lactose; the sugar was coupled to the resin as a p-aminophenylderivative or by means of divinyl sulfone activation. An epoxy group inlinkages of galactose or lactose decreased the binding capacity. Aquantitatively similar degree of protein yields was obtained for thebeta-galactoside-binding protein of bovine heart, although differentproteins were obtained when neoglycoproteins were used as ligand. Thenature of the affinity ligand in lectin purification can increase theyield and may also influence the profile of the carbohydrate-bindingproteins. Author.

Keyword(s): ANIMAL

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