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Glycobiology Advance Access published online on December 23, 2003
Glycobiology, doi:10.1093/glycob/cwh037
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Submitted on October 16, 2003
Revised on November 14, 2003
Accepted on November 18, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis

Patrick Groves 1, Martin Ohsten Rasmussen 2, M. Dolores Molero 3, Eric Samain 2, F. Javier Cañada 1, Hugues Driguez 2, and Jesús Jiménez-Barbero 1*

1 Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
2 Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS), Affiliated with Université Joseph Fourier, BP 53, 38041 Grenoble cedex 9, France
3 Centro de Apoyo a la Investigación, RMN, Facultad de Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain

* To whom correspondence should be addressed. E-mail: jjbarbero{at}cib.csic.es.

Abstract

A series of N-acetyl-chitooligosaccharides (GlcNAc)1-6 have been studied by a NMR method - Diffusion Ordered Spectroscopy (DOSY). DOSY has also been applied to two additional synthetic related oligosaccharides [GlcNH2-(GlcNAc)4 and GlcNH2-(GlcNAc)2-GlcNAcSO3Na]. A plot of the logarithm of the determined diffusion coefficients (logD) of (GlcNAc)n versus the logarithm of molecular weight was linear (6 points, R2=0.995). The molecular weights of the two synthetic chitin derivatives could be estimated to within a 10% error. The processed NMR data of all the chitooligosaccharides was also plotted in a polyacrylamide gel-like format to aid visual interpretation. Moreover, the logD value of the NMR signal resonances of a chitin-binding protein (hevein) changed as a function of a given titrated ligand, (GlcNAc)6. Evidence for a 2:1 hevein:(GlcNAc)6 complex is detected by DOSY at high hevein:(GlcNAc)6 ratios. This data is consistent with published analytical ultracentrifugation and isothermal titration calorimetry data. A 1:1 complex is preferred at higher ligand concentrations. DOSY can complement size exclusion chromatography in carbohydrate research with the advantage that oligosaccharides are more readily detected by NMR.


chitooligosaccharides, diffusion ordered spectroscopy, hevein, protein-carbohydrate interactions, molecular weight standard
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