Skip Navigation


Glycobiology Advance Access originally published online on October 20, 2004
Glycobiology 2005 15(3):303-312; doi:10.1093/glycob/cwi008
This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Data Supplement
Right arrow All Versions of this Article:
15/3/303    most recent
cwi008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (14)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Seyfried, N. T.
Right arrow Articles by Almond, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seyfried, N. T.
Right arrow Articles by Almond, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Glycobiology vol. 15 no. 3 © Oxford University Press 2005; all rights reserved.

Preparation and application of biologically active fluorescent hyaluronan oligosaccharides

Nicholas T. Seyfried2,3, Charles D. Blundell3, Anthony J. Day2,3 and Andrew Almond1,3

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

1 To whom correspondence should be addressed; e-mail: andrew.almond{at}bioch.ox.ac.uk

Received on September 17, 2004; accepted on October 19, 2004

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 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 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein–carbohydrate interactions.

Key words: 2-aminobenzoic acid / electrophoretic mobility shift assay / fluorophore-assisted carbohydrate electrophoresis / mass spectrometry / versican


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
T. Schulz, U. Schumacher, and P. Prehm
Hyaluronan Export by the ABC Transporter MRP5 and Its Modulation by Intracellular cGMP
J. Biol. Chem., July 20, 2007; 282(29): 20999 - 21004.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. D. Blundell, A. Almond, D. J. Mahoney, P. L. DeAngelis, I. D. Campbell, and A. J. Day
Towards a Structure for a TSG-6{middle dot}Hyaluronan Complex by Modeling and NMR Spectroscopy: INSIGHTS INTO OTHER MEMBERS OF THE LINK MODULE SUPERFAMILY
J. Biol. Chem., May 6, 2005; 280(18): 18189 - 18201.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.