Capillary electrophoresis method for the enzymatic assay of galactosyltransferases with postreaction derivatization.
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.
Monegal
Ana
A
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain.
Pinyol
Roser
R
Planas
Antoni
A
D019075:Electrophoresis, Capillary / Q000379:methods*; D005700:Galactosyltransferases / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics
D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase