Resum
Glycosyltransferases are key enzymes in glycoconjugate biosynthesis, which make them important targets for biomedical research. Among the different methodologies developed to analyze glycosyltransferase activities, fluorophore-assisted capillary electrophoresis (FACE) emerges as a powerful technique in carbohydrate analysis. Its application to monitor glycosyltransferase activity has been limited to reactions with derivatized sugars as acceptor substrates in which a charged fluorophore/chromophore must be introduced, thus requiring tedious preparative synthesis and purification for each single acceptor substrate. Here we describe a novel and general glycosyltransferase assay based on FACE using underivatized acceptor substrates. Enzyme activity is monitored by a discontinuous assay with postreaction derivatization by reductive amination with 8-aminonaphthalene-1,3,6-trisulfonic acid. The reaction mixture is directly analyzed by HPCE (high-performance capillary electrophoresis) under inverted electroosmotic conditions at pH 2.5 and 30°C. After method validation, it was applied to the kinetic characterization of an α-1,3-galactosyltransferase, the enzyme responsible for the biosynthesis of αGal epitope involved in the hyperacute rejection in xenotransplantation. The absence of a label on the acceptor during the GT reaction avoids any interference of the label with the enzyme, and the postreaction derivatization does not require any purification step.
Idioma original | Anglès |
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Pàgines (de-a) | 115-123 |
Nombre de pàgines | 9 |
Revista | Analytical Biochemistry |
Volum | 346 |
Número | 1 |
DOIs | |
Estat de la publicació | Publicada - 1 de nov. 2005 |