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.
Hemming
F W
FW
School of Biomedical Sciences, Medical School, University of Nottingham, Nottingham, NG7 2UH, United Kingdom. Frank.Hemming@nottingham.ac.uk
Wallis
G L
GL
Peberdy
J F
JF
D001233:Aspergillus nidulans / Q000201:enzymology; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005069:Evaluation Studies as Topic; D005690:Galactose / Q000031:analogs & derivatives* / Q000032:analysis; D006001:Glycoconjugates / Q000737:chemistry*; D006026:Glycoside Hydrolases; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D008745:Methylation; D008832:Microchemistry / Q000379:methods*; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000032:analysis; D043324:beta-Fructofuranosidase
D006001:Glycoconjugates; C104136:uridine diphosphate galactofuranose; D014530:Uridine Diphosphate; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D005690:Galactose; D000432:Methanol