Study of the enzymatic transformation of fluorescently labeled oligosaccharides in human epidermoid cells using capillary electrophoresis with laser-induced fluorescence detection.
Isomeric oligosaccharides of both beta Gal(1-->3)beta GlcNAc (type I) series and beta Gal(1-->4)beta GlcNAc (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 [beta Gal(1-->3)beta GlcNAc-O-TMR and beta Gal(1-->4)beta GlcNAc-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 (beta Gal(1-->3)beta GlcNAc-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.
Le
X C
XC
Department of Public Health Sciences, Faculty of Medicine, University of Alberta, Edmonton, Canada.
Zhang
Y
Y
Dovichi
N J
NJ
Compston
C A
CA
Palcic
M M
MM
Beever
R J
RJ
Hindsgaul
O
O
D002240:Carbohydrate Sequence; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007834:Lasers; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D009844:Oligosaccharides