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J. Biochem, 1984, Vol. 96, No. 4 1283-1287
© 1984 Japanese Biochemical Society

research-article

High-Performance Liquid Chromatography of Pyridylamino Derivatives of Unsaturated Disaccharides Produced from Chondroitin Sulfate Isomers by Chondroitinases1

Chie KODAMA, Noboru OTOTANI, Mamoru ISEMURA and Zensaku YOSIZAWA

Department of Biochemistry, Tohoku University School of Medicine Sendai, Miyagi 980

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides ({Delta}Di-OS, {Delta}Di-4S, {Delta}Di-6S, and {Delta}Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of µBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/1, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.

1This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by a Research Grant from Takeda Science Foundation.


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