1,4-Butanediol diglycidyl ether coupling of carbohydrates to sepharose: Affinity adsorbents for lectins and glycosidases

Abstract

The use of 1,4-butanediol diglyeidyl ether to couple carbohydrate ligands to Sepharose [Sundberg, L., and Porath, J. (1975) J. Chromatogr. 90, 87; Vretblad, P. (1976) Biochim. Biophys. Acta. 434, 169] has been explored with a number of carbohydrate derivatives, such as amino sugars ( N-acetylglucosamine, N-acetylgalactosamine, nonacetylated short oligomers of glucosamine), methyl glycosides ( O-methyl-α-mannoside and O-methyl-β-galactoside), di- and oligosaccharides (lactose and stachyose), and polysaccharides (yeast mannan and dextrin). Using model reactions it has been established that all but the reducing sugars are sufficiently stable to withstand the alkaline conditions required for the coupling reaction, and that the primary alcohol at the C 6 position of the sugars is the primary site for the coupling reaction. In the case of glucosamine, the amino group is six times more reactive than is the 6-hydroxy group. The following affinity adsorbents have been tested and found useful in the purification of lectins and glycosidases: a yeast mannan adsorbent binds jack bean α-mannosidase (0.15 mg/ml of adsorbent) and concanavalin A (0.5 mg/ml); a dextrin adsorbent binds bovine pancreatic α-amylase (2 mg/ml of adsorbent); a lactose adsorbent binds peanut lectin (5 mg/ml of adsorbent); a (GlcNAc) 3–4 adsorbent binds wheat germ lectin (50 mg/ml of adsorbent); and, as previously reported [Vretblad, P. (1976) Biochim. Biophys. Acta. 434, 169], a GlcNAc adsorbent binds wheat germ lectin (10 mg/ml of adsorbent) and a GalNAc adsorbent binds soybean lectin (18 mg/ml of adsorbent).