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Glycobiology Advance Access originally published online on December 23, 2003
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Glycobiology vol 14 no 5 pp. 451-456, 2004
Glycobiology vol. 14 no. 5 © Oxford University Press 2004; all rights reserved.

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

Patrick Groves2, Martin Ohsten Rasmussen3, M. Dolores Molero4, Eric Samain3, F. Javier Cañada2, Hugues Driguez3 and Jesús Jiménez-Barbero1,2

2 Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain; 3 Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS), Affiliated with Université Joseph Fourier, BP 53, 38041 Grenoble cedex 9, France; and 4 Centro de Apoyo a la Investigación, RMN, Facultad de Quimicas, Universidad Complutense de Madrid, 28040 Madrid, Spain

Received on October 16, 2003; revised on November 14, 2003; accepted on November 18, 2003

A series of N-acetyl-chitooligosaccharides (GlcNAc)1–6 have been studied by a nuclear magnetic resonance (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 log of the determined diffusion coefficients (logD) of (GlcNAc)n versus the log of molecular weight was linear (6 points, R2 = 0.995). The molecular weights of the two synthetic chitin derivatives could be estimated to within 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.

1 To whom correspondence should be addressed; e-mail: jjbarbero{at}cib.csic.es


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