title: Capillary electrophoresis method for the enzymatic assay of galactosyltransferases with postreaction derivatization.
abstract: 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 degrees C. After method validation, it was applied to the kinetic characterization of an alpha-1,3-galactosyltransferase, the enzyme responsible for the biosynthesis of alphaGal 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.
authors.affiliation: Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Monegal
authors.forename: Roser
authors.initials: R
authors.lastname: Pinyol
authors.forename: Antoni
authors.initials: A
authors.lastname: Planas
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D005700:Galactosyltransferases / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics
chemical_list: D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase