Specific Tritium Labeling of beta-D-Galactofuranosides at the 6-Position: A Tool for beta-D-Galactofuranosidase Detection

MARIÑO, KARINA; MARINO, CARLA; LEDERKREMER, ROSA M. DE

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2002 vol. 301 p. 325 - 328

0003-2697

The glycobiology of galactofuranose is attracting considerable interest from many groups, since beta-D-galactofuranosyl residues are constituents of infectious microorganisms, but are absent in mammal glycoconjugates. The enzymes involved in the metabolism of the sugar are good targets for the development of antimicrobial agents. The best known is the specific exo-beta-D-galactofuranosidase first purified from the culture medium of Penicillium fellutanum and later described in Helminthosporium sacchari and Penicillium and Aspergillius species. An affinity-purification method using the inhibitor 4-aminophenyl 1-thio-beta-D-galactofuranoside as ligand for the preparation of the affinity phase was also described. Activity of theenzyme is usually measured with the substrate 4-nitrophenyl beta-D-galactofuranoside. On the other hand the processes involved in Galf incorporation in glycoconjugates have not been completely elucidated. A mutase, which converts UDP-Galp in UDP-Galf with low efficiency (5-8%), has been described. Detection of the galactofuranose intermediates and of theenzymes involved in the metabolism of galactofuranose using radiolabeled precursors and substrates is essential for such studies. Here we report for the first time a procedure for introducing tritium at the 6-position of galactofuranose derivatives, being the key step in oxidationwith pyridinium chlorochromate of HO-6 of a convenient derivative and reduction with NaB3H4. Using the synthesized [3H]methyl-beta-D-galactofuranoside, the activity of beta-D-galactofuranosidase can be followed by detection of radioactive galactose by HPAEC or TLC.