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 specif icity and to detect the most commonly-encountered contaminants in avai lable exoglycosidase preparations (Jacob and Scudder, Methods Enzymol. , 230, 280-300, 1994) were developed. Monosaccharides and oligosacchar ides were analyzed in a single chromatographic step using high-pH anio n-exchange chromatography with pulsed amperometric detection, All anal yses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the ch romatograph, The sialidase from Newcastle disease virus was found to r elease both alpha(2-->3)- and alpha(2-->6)-linked Neu5Ac from a triant ennary, lactosamine-type oligosaccharide, The activity of alpha-galact osidase from green coffee beans was assayed using Gal alpha(1-->3)[Fuc alpha(1ar2)]Gal by detection of Gal and Fuc alpha(1-->3)Gal. The link age specificities of beta-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Gal beta(1-->3 or 4) GlcNAc bet a(1-->3)beta(1-->4)Glc as substrates, Contaminating beta-N-acetylhexos aminidase activity in the beta-galactosidase preparation was assayed u sing an agalactobiantennary oligosaccharide, The alpha(1-->3 or 4) lin kage specificity of fucosidase III from almond meal was confirmed (Scu dder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked alp ha(1-->6). An alpha-fucosidase from chicken liver was found to cleave alpha(1-->2,3 or 6)-linked Fuc residues from oligosaccharides. The act ivity of jack bean (Canavalia ensiformis) alpha-mannosidase was assaye d with a relatively resistant substrate, Man alpha(1-->3)-Man beta(1-- >4)GlcNAc. A GlcNAc beta(1-->4)-terminated triantennary oligosaccharid e was used to assay for contaminating beta-N-acetylhexosaminidase acti vity in alpha-mannosidase preparations and to determine the linkage an d branch specificity of beta-N-acetylhexosaminidase at different enzym e concentrations.