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Glycobiology Advance Access published online on October 20, 2004
Glycobiology, doi:10.1093/glycob/cwi008
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Received September 17, 2004
Accepted October 19, 2004

ORIGINAL ARTICLES

Preparation and application of biologically active fluorescent hyaluronan oligosaccharides

Nicholas T. Seyfried 1, Charles D. Blundell 2, Anthony J. Day 1, and Andrew Almond 2*

1 MRC Immunochemistry Unit, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK; Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
2 Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

* To whom correspondence should be addressed.
Andrew Almond, E-mail: andrew.almond{at}bioch.ox.ac.uk


  Abstract

We report the production of biologically active HA oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from four to 40 residues in length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterization of HA oligosaccharides is facilitated by 2AA derivitization since it enhances signals in MALDI-TOF mass spectrometry and improves FACE (fluorophore-assisted carbohydrate electrophoresis) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labeled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatized oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to the G1-domain of human recombinant versican (VG1). An electrophoretic mobility shift assay was used to study VG1 binding to labeled HA 8-, 10-, 20-, 30- and 40-mers and, although no stable VG1 binding was observed to labeled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA10 was estimated from densitometry analysis of the free oligosaccharide. Interactions involving HA 20-, 30-, and 40-mers (proposed to be multivalent) could also be studied using this protocol. Oligosaccharides labeled with 2-aminobenzoic acid therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

Keywords: 2-aminobenzoic acid (2AA); electrophoretic mobility shift assay (EMSA); mass spectrometry; fluorophore-assisted carbohydrate electrophoresis (FACE); versican.
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