Study of the enzymatic transformation of fluorescently labeled oligosaccharides in human epidermoid cells using capillary electrophoresis with laser-induced fluorescence detection

Abstract

Isomeric oligosaccharides of both βGal(1→3)βGlcNAc (type I) series and βGal(1→4)βGlcNAc (type II) series were studied by using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. A mixture of phenylboronic acid and sodium tetraborate was used in the CE running buffers to improve the electrophoretic separation of the oligosaccharides. Both series of the tetramethylrhodamine (TMR)-labeled substrates [βGal(1→3)βGlcNAc-O-TMR and βGal(1→4)βGlcNAc-O-TMR) and their potential enzymatic products were baseline resolved using CE. The high resolution provided by CE and the excellent detection limit (8·10 −23 mol, or 50 molecules) by LIF allowed for the determination of minor enzyme products in the presence of excess unreacted substrate. The action of competing enzymes acting on the common type I sequence was monitored after the incubation of human epidermoid carcinoma cells (A431) with a fluorescent substrate (βGal(1→3)βGlcNAc-O-TMR). The CE-LIF analyses showed the formation of both synthetic and hydrolytic products, suggesting the actions of glycosyltransferases and glycosidases in the cells.