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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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.

Key words: chitooligosaccharides / diffusion ordered spectroscopy / hevein / molecular weight standard / protein–carbohydrate interactions


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
N-acetyl glucosamine (GlcNAc) is the building block of chitin and Nod factors. Nod factors are secreted from legume roots and signal several responses, including root nodulation involving the invasion of symbiotic rhizobia (Cullimore et al., 2001Go; D'Haeze and Holsters, 2002Go). Nod factors consist of modified ß-1,4-GlcNAc oligosaccharides where the nonreducing end acetyl group has been replaced with a long-chain fatty acid. Other modifications include the reducing end O-6, which can be either fucosylated or sulfonated (D'Haeze and Holsters, 2002Go). For detection, GlcNAc contains rather poor chromophores suitable for UV or fluorescence detection. Consequently, the analyses of synthetic or naturally occurring chitooligosaccharides are often carried out by means of indirect and/or complex protocols. It is well known that nuclear magnetic resonance (NMR) may be the method of choice for elucidating oligo- and polysaccharide structures. In particular, 1H NMR spectroscopy adequately characterizes carbohydrate chains, such as those of chitooligosaccharides, because the monosaccharide residues, GlcNAc, contain a number of detectable protons. Only a few seconds are required to acquire a good-quality spectrum of submillimolar-concentrated oligosaccharide on a modern instrument. Within minutes, spectra of samples in the micromolar range can be obtained. However, it has to be considered that saccharide impurities are likely to provide additional or overlapping resonances that might be difficult to characterize.

Diffusion ordered spectroscopy (DOSY) (Stilbs, 1981Go) provides a method of molecular size determination through the measurement of diffusion coefficients (logD). This NMR method has found a number of applications. In the study of drug-receptor interactions (Cameron and Fielding, 2002Go), the logD values of drugs were measured in the presence and absence of a small amount of receptor. The measured logD values were a weighted average of receptor-bound and free ligand. Therefore, ligands whose NMR signals increased in apparent molecular weight (MW) signified a receptor-bound population, whereas ligands that did not bind to the receptor experienced no change in logD. DOSY is also useful in resolving chemical mixtures and can provide the 1D 1H NMR spectra and the logD of individual components (Diaz and Berger, 2000Go; Pelta et al., 2002Go).

The measurement of accurate diffusion coefficients is prone to several potential errors. The gradients must be correctly calibrated, and temperature convection inside the NMR tube and sample-induced changes in viscosity must be avoided (Sorland and Aksnes, 2002Go). Herein, we report a simple strategy to overcome these drawbacks using standard equipment based on similar protocols to those developed for the pharmaceutical industry (Kerssebaum, 2002Go). We also produce novel representations of DOSY data that are more readily interpreted.

Hevein is a small chitin-binding protein. The resolution of the 3D structure of hevein and its interactions with N-acetyl-chitooligosaccharides has been the focus of our attention and of others (Asensio et al., 1995Go, 1998Go, 2000aGo,bGo; Siebert et al., 2000Go) because it provides a model for the study of carbohydrate–protein interactions. The NMR structures of a variety of hevein complexes suggest that these protein domains efficiently bind N-acetyl-chitooligosaccharides, with (GlcNAc)3 moieties providing the basic interactions for the molecular recognition processes (Asensio et al., 2000aGo,bGo). Moreover, the recognition process is multivalent, because analytical ultracentrifugation measurements have indicated that two hevein molecules can bind to (GlcNAc)8, particularly when the hevein concentration exceeds the (GlcNAc)8 concentration (Asensio et al., 2000aGo). In addition, isothermal microcalorimetry data for hevein binding to (GlcNAc)5 could not be fitted to either a 1:1 or 2:1 model (Asensio et al., 2000aGo), suggesting a mixture of 1:1 and 2:1 complex species.

We have chosen the system provided by (GlcNAc)n and hevein to illustrate the application of DOSY in characterizing the size of different N-acetyl-chitooligosaccharides and their complexes to protein receptors. Thus the stoichiometry of the complexes formed by hevein with (GlcNAc)6 have been evaluated by DOSY as a complementary method to other currently used physical biochemical methods, such as analytical ultracentrifugation and size exclusion chromatography (SEC).


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Chitooligosaccharides 1 and 2 (Figure 1) were produced in high yield from an Escherichia coli expression system (Samain et al., 1997Go, 1999Go). After purification by silica gel chromatography, the two chitooligosaccharides were characterized by 1D and 2D1H and 13C-NMR experiments and by mass spectrometry. Although these spectra proved the identity of the desired products, none were able to prove their purity without derivatization. The unambiguous demonstration of purity in saccharide molecules is a difficult task. Figures 2A and 2B provide a typical representations of 2D DOSY spectra of 1 and 2. The diffusion coefficient is a property of the molecule as a whole and therefore is shared by all the proton resonances of the molecule. Therefore, the signals of 1 and 2 are spread out in a horizontal line with average logD values of –9.610 and –9.570, respectively. The solvent peak can be seen at 4.75 ppm and logD = –8.716. If impurities of different MW were present in the samples, then a second set of peaks, with a different logD value, would also be observed. Overlapped signals are expected to provide misplaced or distorted peaks, as is the case for one anomeric 1H signal that overlaps with part of the solvent signal (asterisked peaks in Figures 2A and 2B). Thus Figures 2A and 2B show that both 1 and 2 are essentially free of impurities with different MW.



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Fig. 1. Chemical structures of chitooligosaccharides GlcNH2-(GlcNAc)4 (1) and GlcNH2-(GlcNAc)2-GlcNAcSO4 (2).

 



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Fig. 2. DOSY analysis of chitooligosaccharides. (A) 2D DOSY spectrum of 1. (B) 2D DOSY spectrum of 2. The x-axis contains the standard 1H dimension, and the y-axis contains the diffusion dimension. The area bound by the bold boxes in A and B are reproduced in Figure 3B (lanes 7 and 8, respectively).

 



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Fig. 3. Alternative presentations of DOSY data. (A) Plot of logD versus logMW for a series of N-acetyl-chitooligosaccharides, (GlcNAc)1–6 (left to right, circles). A least-squares linear correlation was fitted to the data to yield the fit: logD = –0.446 logMW –8.270, r2 = 0.995. The square symbols denote the constant diffusion coefficient measured for the solvent signal. (B) Partial DOSY spectra corresponding to the sugar ring proton region (3.4–3.9 ppm) and logD between –9.2 and –9.8, also corresponding to the bold boxed regions of Figure 2A and 2B. Lanes 1–6 correspond to (GlcNAc)1–6, lane 7 to 1, and lane 8 to 2.

 
The DOSY spectra in Figure 2 only provides information about the purity of 1 and 2 but not the molecular size, as the calibration of gradients, temperature, and sample viscosity effects were not fully accounted for in this example. Because an accurate determination of logD requires a rigorous treatment of these factors, we explored an alternative method of obtaining reliable molecular size data. Figure 3A presents the logD value of a series of homologous N-acetyl-chitooligosaccharides, (GlcNAc)n, which differ in length/size (n = 1–6), against their logMW, used as calibration curve. The interpolation of the logD values for 1 and 2 as derived from their DOSY data suggest that the mass of 1 is 1015 Da, whereas that of 2 is 827 Da. These results are within 10% of the calculated masses of 992 Da for 1 and 891 Da for 2. The logD value for the solvent is constant for all of the samples, Figure 3A, and this signal provides an internal standard that indicates the viscosity of the samples does not change.

Figure 3B provides an alternative representation of the data presented in Figure 3A. Narrow strips from the 2D DOSY spectrum (as indicated by the bold boxes in Figures 2A and 2B) are taken and displayed side by side. The resulting figure can be interpreted the same way as the bands/spots obtained in a polyacrylamide gel. Smaller oligosaccharides are found at the bottom, and larger saccharides toward the top of the strips. The DOSY results of 1 and 2 are also presented in Figure 3B, lanes 7 and 8, respectively. It can be quickly and readily deduced that these molecules contain the correct number of sugar units by comparison to the other strips in the panel.

Passing to saccharide binding to receptors and choosing hevein as a model, Figure 4A shows fluorescence spectroscopy data for hevein binding to (GlcNAc)6 that can be fitted to an equation assuming 1:1 stoichiometry when determined at low, micromolar protein concentration (K = 6.0 ± 1.0 x 105 M–1). However, it has been demonstrated that multivalent effects may take place for (GlcNAc)5 and (GlcNAc)8 at millimolar concentrations (Asensio et al., 2000aGo). Therefore we also decided to employ DOSY as an alternative to determine the stoichiometry of the hevein:(GlcNAc)6 complex. Six hevein resonances, which do not overlap with ligand resonances, were followed by DOSY during a titration with (GlcNAc)6. Changes in logD are plotted against the protein:ligand stoichiometry. In this case, a calibration curve formed by five proteins of MW between 6.5 and 66 kDa (not shown, logD = –0.427 logMW –8.231, r2 = 0.983) was used. A hevein concentration of 0.3 mM, together with the measured binding constant from the fluorescence experiments, means that stoichiometric binding of (GlcNAc)6 should be observed as indicated by the theoretical plot given in Figure 4B. The expected values of the 1:1 and 2:1 protein:ligand complexes are also indicated to the right of Figure 4B. Titration of up to 0.5 equivalents of ligand results in a sharp increase in the logD values in excess of that expected for 1:1 stoichiometry, approaching that for 2:1 stoichiometry. The titration between 0.5 and 1.0 equivalents of ligand results in a decrease in logD values back toward that expected for a 1:1 complex. The logD value is constant between 1.0 and 3.0 equivalents of added ligand and slightly elevated compared to that expected for a 1:1 complex. Therefore the DOSY experiment for this system easily allows the detection of a significant proportion of 2:1 protein:ligand complex for protein:ligand ratios higher than 1:1.




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Fig. 4. Titration data for (GlcNAc)6 added to hevein. (A) The change in the tryptophan emission fluorescence of 1 µM hevein is plotted as a function of added (GlcNAc)6. A least squares fitted line for 1:1 ligand:host interactions is fitted to the data and yields a K = 6.0 ± 1.0 x 105 M–1. (B) Plot of the change in logD ({Delta}logD) as a function of stoichiometrically added (GlcNAc)6 to a 300 µM hevein solution. The predicted {Delta}logD values for free hevein (1:0), as well as 1:1 and 2:1 protein:oligosaccharide complexes, are indicated to the right of the panel. The dotted line indicates the theoretical logD values that should be obtained for the formation of a 1:1 hevein:(GlcNAc)6 complex.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
DOSY is a 2D experiment, collected as a series of 16–64 1D 1H experiments. Generally speaking it requires less time to acquire DOSY data than standard 2D NMR experiments, such as correlation spectroscopy and nuclear Overhauser effect spectroscopy. Therefore we recommend acquiring DOSY data during the collection of routine NMR experiments to provide additional complementary data about the purity of an oligosaccharide sample. DOSY has been described as "in tube chromatography" (Johnson, 1999Go) and has value in solving inconsistencies arising between thin-layer chromatography results and standard NMR analyses.

The Stokes-Einstein equation that describes diffusion phenomena and from which the analysis of DOSY data is derived, holds for spherical molecules. Therefore it is interesting that Figure 3A yields a linear correlation, even for relatively long N-acetyl-chitooligosaccharides, which are expected to adopt nonspherical structures (González et al., 1999Go). Such a linear correlation of logD versus logMW has also been observed with ß-amyloid-derived polypeptides (Danielsson et al., 2002Go), proteins (Nesmelova et al., 2002Go; Wilkins et al., 1999Go), as well as cyclodextrins and their inclusion complexes (Cameron and Fielding, 2002Go).

Figure 3A can be compared with SEC calibration plots of logVe/V0 against logMW generated from the relative mobility of MW standards. These plots circumvent systematic errors that otherwise affect size determination by SEC (accurate determination of pump rates, total and excluded column volumes). In the same way, Figure 3A may surmount some of the systematic errors associated with DOSY (gradient calibration, temperature convection) (Sorland and Aksnes, 2002Go), even if some of these errors, such as gradient calibration (Damberg et al., 2001Go), can be significantly corrected. Access to a series of compounds allows a calibration curve be easily constructed using the same solvent or buffer as for the sample. However, as it has been found that the relationship between logD and logMW for a series of proteins depends on whether the proteins are folded or denatured (Wilkins et al., 1999Go), it is clear that the choice of reference compounds is important. Nonetheless, the reference curve allows a more reliable MW to be obtained for the unknown. In principle, the calculated MW should give a consistent value when determined on any NMR instrument that provides a good calibration plot with suitable reference compounds.

The advantages of DOSY over SEC in the carbohydrate field include an ease of detection (all carbohydrates contain protons that can be detected by NMR), compatibility with a wide range of solvents, and the fact that experiments are essentially performed under equilibrium conditions. Disadvantages include the existence of overlapping NMR resonances—particularly a concern for saccharide mixtures, that might have similar proton signals. Nevertheless, analytical algorithms are being improved (Armstrong et al., 2003Go; Mouro et al., 2002Go) and extension to 3D NMR DOSY experiments may also alleviate the overlapping problem.

The representation of DOSY data need not be as shown in Figure 2, although this is the best representation to confirm the absence of overlapping signals from solvents or chemicals used in synthesis. With Figure 3A, the quality of the NMR data cannot be evaluated. For this reason, Figure 3B might be favored, because it provides part of the NMR data that can be easily evaluated by readers. Calculated MWs can be quoted as based on the linear fit provided by Figure 3A.

Multiple hevein binding to oligosaccharides is not directly detected by fluorescence. This might be due to the relatively low affinity of the ligand for the protein at the 1 µM protein concentration used in the titrations and the fact that only 2 of the 18 titration points are collected below a 2:1 protein:(GlcNAc)6 stoichiometry. The fact that the a full 2:1 stoichiometry is not observed in Figure 4B and that the peak at 2:1 stoichiometry is symmetrical in the DOSY experiment suggests that the hexasaccharide provides two binding sites, in a case of multivalency, but with either a lack of cooperativity or even with steric hindrance between the two sites. It is also possible that hevein has a preference to bind to the middle portion of the hexasaccharide, as also supported by earlier studies (Asensio et al., 2000aGo), which would hinder the binding of further protein molecules to the available mono- or disaccharide ends of the hexasaccharide.

To summarize, DOSY is a powerful NMR experiment that can help characterize an oligosaccharide by confirming the absence of small chemicals used in synthesis and, when measured against appropriated mass standards, its correct molecular size (to a practical limit of octasaccharide, Figure 3B). The calculated masses of chitooligosaccharides 1 and 2 are within 10% of their calculated values, which were confirmed by mass spectrometry. Because we applied a readily available protocol (Kerssebaum, 2002Go) without implementing improvements (Armstrong et al., 2003Go; Damberg et al., 2001Go; Mouro et al., 2002Go), it should be possible to improve the accuracy of DOSY measurements. DOSY might be employed as a complementary tool to SEC in studying intermolecular interactions, as demonstrated here for the hevein:(GlcNAc)6 complex.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
(GlcNAc)2–6 were purchased from Toronto Research Chemicals (Toronto, Canada). Protein size standards, aprotonin (6.5 kDa), {alpha}-lactalbumin (14 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), and bovine serum albumin (66 kDa), were purchased from Sigma (St. Louis, MO). Hevein was purified as described in Asensio et al. (1995Go, 1998Go). All other chemicals were from Aldrich (Milwaukee, WI).

Chitooligosaccharide expression and purification
Chitooligosaccharides 1 and 2 were obtained from metabolically engineered E. coli, as described (Samain et al., 1997Go, 1999Go). Both compounds were further purified by silica gel chromatography. Compound 1 was purified on Merck Silica gel 40–63 µm with a acetonitrile/water/ammonia gradient (70:30:1 to 60:40:1) with 89% recovery of the material. The mass spectrum (matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF]) of 1 gave m/z 1014 (M + Na+). Compound 2 (CO-IV(S, NH2) was purified on Merck Silica gel 63–200 µm with a chloroform/methanol/water gradient (40:30:1 to 35:40:5) with 33% recovery of the material. The mass spectrum (MALDI-TOF) of 2 gave m/z 913 (M + Na+). MALDI-TOF measurements were performed on a Bruker Daltonics Autoflex apparatus.

NMR spectroscopy
All samples were prepared (~1 mM concentration) in 20 mM phosphate buffer, 100 mM NaCl, pH 5.6, 100% D2O. The standard Bruker DOSY protocol was used at 298 K on an Advance 500 MHz (Kerssebaum, 2002Go) equipped with a broad-band z-gradient probe. Thirty-two 1D 1H spectra were collected with a gradient duration of {delta} = 2 ms and an echo delay of {Delta} = 100 ms for oligosaccharides, or 250 ms for the hevein complexes. Acquisition times of 8 and 32 min were required for the oligosaccharide samples and hevein complexes, respectively. Samples of aprotonin (6.5 kDa), {alpha}-lactalbumin (14 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), and bovine serum albumin were used to calibrate the hevein/(GlcNAc)6 titration (logD = –0.427 logMW –8.231, r2 = 0.983) in a similar way as described for Figure 3A. The ledbpg2 s pulse sequence, with stimulated echo, longitudinal eddy current compensation, bipolar gradient pulses, and two spoil gradients, was run with a linear gradient (53.5 G cm–1) stepped between 2% and 95%. The 1D 1H spectra were processed and automatically baseline corrected. The diffusion dimension, zero-filled to 1 k, was exponentially fitted according to preset windows for the diffusion dimension (–8.5 < logD < –10.0 [oligosaccharides] or –10.5 [hevein complexes]).

Fluorescence spectroscopy
Fluorescence spectroscopy was performed on a Hitachi F-2500 spectrometer. Experiments were performed in a 20 mM phosphate, 100 mM NaCl, pH 5.6 buffer at 25.0°C maintained by a Julaba F-12 temperature control unit. Data were collected with 1 scan at 1 nm/s with excitation and emission slits of 5 nm. The maximum change in hevein emission fluorescence (335 nm) was followed as a function of added (GlcNAc)6 after excitation at 280 nm. The binding curve was least squares fitted to an equation assuming 1:1 ligand:host interactions using SigmaPlot (SPSS, Chicago, Il).


    Acknowledgements
 
We thank the EU for financial support through the SACCSIGNET project (HPRN-CT-2002-00251). The Madrid team also acknowledges Dirección General de Investigación of Spain, Grant BQU2003-03550-C03-01 for funding. CAI of NMR of the Universidad Complutense de Madrid is acknowledged for providing the spectrometer time.


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


    Abbreviations
 
DOSY, diffusion ordered spectroscopy; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MW, molecular weight (Da); NMR, nuclear magnetic resonance; SEC, size exclusion chromatography


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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