Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis.
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 [GlcNH(2)-(GlcNAc)(4) and GlcNH(2)-(GlcNAc)(2)-GlcNAcSO(3)Na]. A plot of the log of the determined diffusion coefficients (logD) of (GlcNAc)(n) versus the log of molecular weight was linear (6 points, R(2) = 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.
Groves
Patrick
P
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Rasmussen
Martin Ohsten
MO
Molero
M Dolores
MD
Samain
Eric
E
Cañada
F Javier
FJ
Driguez
Hugues
H
Jiménez-Barbero
Jesús
J
D000818:Animals; D002686:Chitin / Q000737:chemistry*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000737:chemistry; D013057:Spectrum Analysis / Q000379:methods*
D009844:Oligosaccharides; D002686:Chitin