CHARACTERIZATION OF 9-AMINOPYRENE-1,4,6-TRISULFONATE-DERIVATIZED SUGARS BY CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION

Mono- and oligosaccharides with reducing ends were derivatized with 9- aminopyrene-1,4,6-trisulfonate (APTS) by reductive amination. The resu lting adducts were characterized by capillary electrophoresis (CE) wit h laser-induced fluorescence (LIF) detection. The APTS-derivatized sug ars have significant absorption at 488 nm (35% of its 455 mm maximum a bsorption), while APTS (lambda(max) = 424 mn) has only 4% of its lambd a(max) absorption. When excited at 488 mm, the APTS-derivatized sugars fluoresce at a maximum wavelength of 512 mm, while APTS itself has a much weaker fluorescence with a maximum at 501 nm. By using a 488-nm a rgon-ion laser for excitation and a narrow band filter at 520 nm for f luorescence emission, the APTS-derivatized sugars can be selectively d etected while the signal of the excess APTS reagent is drastically sup pressed. The APTS-derivatized monosaccharides were readily separated i n berate buffer (pH 10.2), while the oligosaccharide ladders were anal yzed using either an acidic phosphate buffer (pH 2.2) or the alkaline borate buffer for separation. The present APTS derivatization chemistr y is capable of converting 2 pmol of sugar analyte to the fluorescent derivative detectable by the present CW LIF procedure. The use of CE/L IF as a tool for the investigation of the specificity of enzyme action of glycosidases using an end-labeled oligosacchariae substrate is als o demonstrated.