Preparation and application of biologically active fluorescent hyaluronan oligosaccharides.
Identifieur interne : 002363 ( PubMed/Corpus ); précédent : 002362; suivant : 002364Preparation and application of biologically active fluorescent hyaluronan oligosaccharides.
Auteurs : Nicholas T. Seyfried ; Charles D. Blundell ; Anthony J. Day ; Andrew AlmondSource :
- Glycobiology [ 0959-6658 ] ; 2005.
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : Fluorescent Dyes, Hyaluronic Acid.
- chemical , metabolism : Hyaluronic Acid.
- chemical , pharmacology : Hyaluronic Acid.
- drug effects : Protein Binding.
- Animals, Cell Line, Drosophila, Sheep, Spectrometry, Fluorescence, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization.
Abstract
We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from 4 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 derivatization because it enhances signals in MALDI-TOF MS 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 HA 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 HA(10) 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 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.
DOI: 10.1093/glycob/cwi008
PubMed: 15496500
Links to Exploration step
pubmed:15496500Le document en format XML
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<author><name sortKey="Seyfried, Nicholas T" sort="Seyfried, Nicholas T" uniqKey="Seyfried N" first="Nicholas T" last="Seyfried">Nicholas T. Seyfried</name>
<affiliation><nlm:affiliation>MRC Immunochemistry Unit, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.</nlm:affiliation>
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</author>
<author><name sortKey="Blundell, Charles D" sort="Blundell, Charles D" uniqKey="Blundell C" first="Charles D" last="Blundell">Charles D. Blundell</name>
</author>
<author><name sortKey="Day, Anthony J" sort="Day, Anthony J" uniqKey="Day A" first="Anthony J" last="Day">Anthony J. Day</name>
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<author><name sortKey="Almond, Andrew" sort="Almond, Andrew" uniqKey="Almond A" first="Andrew" last="Almond">Andrew Almond</name>
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<sourceDesc><biblStruct><analytic><title xml:lang="en">Preparation and application of biologically active fluorescent hyaluronan oligosaccharides.</title>
<author><name sortKey="Seyfried, Nicholas T" sort="Seyfried, Nicholas T" uniqKey="Seyfried N" first="Nicholas T" last="Seyfried">Nicholas T. Seyfried</name>
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<term>Cell Line</term>
<term>Drosophila</term>
<term>Fluorescent Dyes (chemistry)</term>
<term>Hyaluronic Acid (chemistry)</term>
<term>Hyaluronic Acid (metabolism)</term>
<term>Hyaluronic Acid (pharmacology)</term>
<term>Protein Binding (drug effects)</term>
<term>Sheep</term>
<term>Spectrometry, Fluorescence</term>
<term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Fluorescent Dyes</term>
<term>Hyaluronic Acid</term>
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<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Hyaluronic Acid</term>
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<keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Hyaluronic Acid</term>
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<keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>Protein Binding</term>
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<keywords scheme="MESH" xml:lang="en"><term>Animals</term>
<term>Cell Line</term>
<term>Drosophila</term>
<term>Sheep</term>
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<front><div type="abstract" xml:lang="en">We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from 4 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 derivatization because it enhances signals in MALDI-TOF MS 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 HA 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 HA(10) 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 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.</div>
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<Abstract><AbstractText>We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from 4 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 derivatization because it enhances signals in MALDI-TOF MS 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 HA 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 HA(10) 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 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.</AbstractText>
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