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Glycobiology vol 6 no 8 pp. 823-829, 1996
© 1996

research-article

Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine

Anna H.K. Plaas1,3, Vincent C. Hascall2 and Ronald J. Midura2

1Shriners Hospital for Crippled Children, Tampa Unit Department of Biochemistry and Moleclular Biology, University of South Florida Medical School Tampa, FL 33612, USA
2Shriners Hospital for Crippled Children, Tampa Unit Department of Biomedical Engineering, Research Institute of the Cleveland Clinic Foundation Cleveland, OH 44195, USA

3To whom correspondence should be addressed at: Cell Biology Laboratory, Shnners Hospital for Crippled Children, 12502 North Pine Drive, Tampa, FL 33612

Received on May 14, 1996; revised on July 2, 1996; accepted on July 5, 1996

Sulfated glycosaminoglycans such as chondroitin sulfate are composed of three structural domains, a linkage oligosaccharide, connecting the chain to the core protein, a variably sulfated disaccharide repeat structure within the chain and a nonreducing terminal, and these domains may confer specific functions on particular chain populations. We report here a new and highly sensitive method for the detection and quantitation of all nonreducing terminal residues and internal disaccharides obtained by chondroltinase ABC or ACII digestion of aggrecan chondroitin sulfate. The procedure involves a quantitative reductive amination of the reducing ends of sulfated mono- and disaccharide chondroitinase products with 2-aminopyridine and boranedimethylamine. All derivatized saccharides can be separated and quantitated by fluorescence in a single chromatographic step on an AS4A anion exchange column, eluted with a gradient (0–500 mM) of sodium trifluoroacetate. The reproducibility and stability of the derivatisation, together with the sensitivity of the chromatography system, allowed for routine quantitation in the range of 3–500 pmol of reducing group (corresponding to about 1.5–250 ng of disaccharide or 0.75–125 ng of monosaccharide). Moreover, the fluorescence yield (fluorescence area units per pmol of reducing group) was virtually identical for all saccharides analyzed. Application of this method to an analysis of aggrecan purified from calf epiphyseal cartilage and from rat chondrosarcoma chondrocyte cultures allowed a precise identification and quantitation of the internal disaccharides and the nonreducing terminal structures, together with an estimation of the number average molecular weight of CS chains in these aggrecan preparations.

aggrecan chondroitin sulfate glycosaminoglycans proteoglycans


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