Exoglycosidase purity and linkage specificity: Assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection

Simplified HPLC protocols to determine the activity and linkage specificity and to detect the most commonly-encountered contaminants in available exoglycosidase preparations (Jacob and Scudder, Methods Enzymol., 230, 280-300, 1994) were developed. Monosaccharides and oligosaccharides were analyzed in a single chromatographic step using high-pH anion-exchange chromatography with pulsed amperometric detection. All analyses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the chromatograph. The sialidase from Newcastle disease virus was found to release both α(2→3)- and α(2→6)-linked Neu5Ac from a triantennary, lactosamine-type oligosaccharide. The activity of α-galactosidase from green coffee beans was assayed using Galα(1→3)[Fucα(1ar2)]Gal by detection of Gal and Fucα(1→3)Gal. The linkage specificities of β-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Ga1β(1→3 or 4)GlcNAcβ(1→3)β(1→4)Glc as substrates. Contaminating β-N-acetylhexosaminidase activity in the β-galactosidase preparation was assayed using an agalactobiantennary oligosaccharide. The α(1→3 or 4) linkage specificity of fucosidase III from almond meal was confirmed (Scudder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked α(1→6). An α-fucosidase from chicken liver was found to cleave α(1→2,3 or 6)-linked Fuc residues from oligosaccharides. The activity of jack bean (Canavalia ensiformis) α-mannosidase was assayed with a relatively resistant substrate, Manα(1→3)-Manβ(1→4)GlcNAc. A GlcNAcβ(1→4)-terminated triantennary oligosaccharide was used to assay for contaminating β-N-acetylhexosaminidase activity in α-mannosidase preparations and to determine the linkage I and branch specificity of β-N-acetylhexosaminidase at different enzyme concentrations.