A pyridylamination method aimed at automatic oligosaccharide analysis of N-linked sugar chains. |
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Authors: | K Yanagida S Natsuka S Hase |
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Affiliation: | Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka, 560-0043, Japan. |
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Abstract: | The procedure for preparation of pyridylaminated sugar chains from glycoproteins was improved with a view to its eventual automation. Following on the coupling reaction improvement already reported [N. Kuraya and S. Hase (1992) J. Biochem. 112, 122-126], two further aspects were improved in this study. Instead of sodium bicarbonate-acetic anhydride, volatile reagents were adopted for the re-N-acetylation of hexosamine residues after hydrazinolysis to give rapid removal of excess reagents. Subsequent to the pyridylamination reaction, excess reagents were removed by cation-exchange to isolate the pyridylaminated oligosaccharides in place of gel filtration. These alterations rendered a one-pot reaction possible and resulted in a large reduction in the amount of time needed compared with other methods so far reported. The procedure was successfully applied to the detection of sugar chains from Taka-amylase A and human erythrocyte membranes. |
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