An unambiguous microassay of galactofuranose residues in glycoconjugates using mild methanolysis and high pH anion-exchange chromatography

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 degrees C followed by high pH anion-exchange chromatography using a routine monosaccharide system. The methanolysis products Mealpha-Galf and Mebeta-Galf were characterized chromatographically by comparison with the authentic compounds and by their response to treatment with mild acid and with beta-galactofuranosidase. Testing against p-nitrophenyl-beta-Galf and UDPalpha-Galf showed the method to be applicable to both alpha- and beta- 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 alpha- and beta-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 beta-configuration. As expected, when applied to asialofetuin (known to contain galactose only in the pyranoside form, Galp), NPalpha-Galp, NPbeta-Galp, or UDPalpha-Galp, mild acid methanolysis failed to produce any galactofuranoside.