SEPARATION OF YEAST ASPARAGINE-LINKED OLIGOSACCHARIDES BY HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY

Oligosaccharides obtained from Saccharomyces cerevisiae mannoproteins by digestion with endo-N-acetyl-β-d-glucosaminidase H were fractionated by anion-exchange chromatography, by elution with 50-100mM NaOH without or with a sodium-acetate gradient, and detected with a pulsed amperometric detector (PAD). The elution times of homologous oligosaccharides fell on a straight line having a slope characteristic of the structural type. The response of the PAD detector per mole of oligosaccharide increased about 2-fold going from Man3GlcNAc to Man13GlcNAc, and appeared to depend primarily on the oxidation of the reducing-end N-acetylglucosamine unit common to all the oligosaccharides. The digestion of a Man10GlcNAc with jack-bean a-mannosidase was monitored by injecting portions of the crude reaction mixture, and the intermediates were characterized by their elution positions and n.m.r. spectra in the anomeric proton region. One commercial jack-bean a-mannosidase preparation contained a novel endolytic activity that released N-acetylglucosamine from the reducing ends of the oligosaccharides and was shown to convert P→6aMan→6aMan→6βMan→4aβGlcNAc to P→6aMan→6aMan→6aβMan plus free N-acetylglucosamine. Another commercial jack-bean a-mannosidase converted the Man10GlcNAc to a Man3GlcNAc having the structure aMan→6βMan→4aβGlcNAc, {A figure is presented} whereas the Oerskovia sp.a-mannosidase converted the same oligosaccharide to a Man4GlcNAc having the structure {A figure is presented}. © 1990.