Rapid assay of beta-galactosidase and sialyltransferase by lectin affinity high performance liquid chromatography with fluorescence detection.
Fluorescein isothiocyanate (FITC)-labelled asialotransferrin and pyridyl aminated oligosaccharides were prepared from asialotransferrin and human milk using affinity chromatography and high performance liquid chromatography (HPLC), respectively. These substances were incubated with galactosidase or sialyltransferase and then examined by lectin affinity HPLC. The elution patterns changed according to the period of incubation and amount of enzyme. This analytical method using lectin affinity HPLC with fluorescence labelled glycoprotein or oligosaccharides as the substrates has great value for detecting these enzyme under the same chromatographic conditions. In addition, differences were noted in the activity of beta-galactosidase toward oligosaccharides having the Gal beta(1----3)GlcNAc or Gal beta(1----4)GlcNAc structure at reducing termini.
Harada
H
H
Hohnen Oil Co., Ltd., Tokyo, Japan.
Ueno
Y
Y
Kamei
M
M
Ohura
R
R
Tanabe
N
N
Uchida
Y
Y
Koyama
F
F
Yamaguchi
T
T
D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005696:Galactosidases / Q000032:analysis*; D007700:Kinetics; D037102:Lectins; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D037121:Plant Lectins; D010944:Plants / Q000201:enzymology; D012799:Sialyltransferases / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013861:Thiocyanates; D001616:beta-Galactosidase / Q000032:analysis*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D037102:Lectins; D037121:Plant Lectins; D013861:Thiocyanates; D012799:Sialyltransferases; D005696:Galactosidases; D001616:beta-Galactosidase; D016650:Fluorescein-5-isothiocyanate