Analyses of Oligosaccharides by Tagging the Reducing End with a Fluorescent Compound

Abstract

The fluorescent oligosaccharide derivative having a positive charge described in the preceding paper (Hase et al. (1979) J. Biochem. 85, 217–220) was shown to be useful for the purpose of determining the sequence and the linkage points of the sugar units. In the sequence analyses of an oligosaccharide, the 2-aminopyridine derivative was partially hydrolyzed. The 2-amino-pyridine derivatives in the hydrolysates were separated by paper electrophoresis (pH 5.0). Each derivative (a monosaccharide derivative to an oligosaccharide derivative) was eluted and the component sugars were analyzed by TLC. In order to determine the linkage points, 2-aminopyridine derivative of an oligosaccharide was permethylated and then partially hydrolyzed. Newly produced hydroxyl groups were methylated with [²H₃] The neutral and basic components were separated by passage through a Dowex 50 (H⁺) column, the fluorescent components having [²H₃] groups were separated by TLC, and each was eluted and hydrolyzed. The hydrolysates were converted into alditol acetates. The linkage points of the sugars were judged by determining the position of [²H₃]methyl groups in the tetramethyl alditol acetates by gas chromatography-mass spectrometry. The method, which requires no quantitative manipulation; was applied to some known oligosaccharides, lacto-N-tetraose, a tetrasaccharide isolated from a glycoprotein(fetuin), and maltotriose.