Enzymatic assay of galactosyltransferase by capillary electrophoresis.

The kinetic parameters of a galactosyltransferase-catalyzed reaction were determined for the first time using capillary zone electrophoresis (CZE) using the methylumbelliferyl (MU) glycoside of N-acetylglucosamine as the acceptor molecule. The CZE was performed using borate buffer and the enzymatic transformations were monitored at 214 nm. The kinetic parameters obtained for MU-GlcNAc were Km = 35.9 microM and Vmax = 7.5 micromol/min/mg, and those for UDP-Gal were Km = 115.3 microM and Vmax = 12.4 micromol/min/mg. A representative inhibition assay was also carried out using UDP as an inhibitor to give the Ki value of 83.9 microM against MU-GlcNAc. The structure of the synthetic product was also confirmed using 1H NMR spectroscopies after isolation by simple chromatography. Copyright 1998 Academic Press.