An Unambiguous Microassay of Galactofuranose Residues in Glycoconjugates Using Mild Methanolysis and High pH Anion-Exchange Chromatography

Abstract

An original, unambiguous microassay of galactofuranose (Galf) residues in glycoconjugates is described. The method involves mild acid methanolysis (5 mM HCl) for 3 h at 84°C followed by high pH anion-exchange chromatography using a routine monosaccharide system. The methanolysis products Meα-Galf and Meβ-Galf were characterized chromatographically by comparison with the authentic compounds and by their response to treatment with mild acid and with β-galactofuranosidase. Testing against p-nitrophenyl-β-Galf and UDPα-Galf showed the method to be applicable to both α- and β- galactofuranosides over the range 10–200 pmol. The results of partial mild methanolysis over shorter periods were consistent with initial inversion of anomeric configuration at methylation followed by anomerization to an equilibrium mixture of α- and β-forms. When applied to a sample of invertase from Aspergillus nidulans, the method indicated that all of the mild acid-labile galactose (78% of the total galactose present) was in the form of a galactofuranoside and that much of this was in the β-configuration. As expected, when applied to asialofetuin (known to contain galactose only in the pyranoside form, Galp), NPα-Galp, NPβ-Galp, or UDPα-Galp, mild acid methanolysis failed to produce any galactofuranoside.