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A convenient method for the structural determination of glycans related to diseases
International Congress Series 1223 (2001) 151 – 159
A convenient method for the structural determination of glycans related to diseases Ikuko Ishii-Karakasa* Department of Biochemistry, School of Medicine, Kitasato University, Kitasato, Sagamihara, Kanagawa, Japan
Abstract A new analytical method for the structural determination of a small quantity of cell surface glycans related to diseases was developed. Oligosaccharides liberated from cell surface glycans were pyridylaminated, and pyridylamino oligosaccharides were separated and purified by high performance liquid chromatography (HPLC). The fraction presumed to be a pyridylamino tetrasaccharide on HPLC analysis was determined to be NeuAca2-3Galb1-3 (NeuAca2-6) GalNAc-PA (I-PA) by the 600-MHz 1H-NMR spectra using the following method. Since the 1HNMR spectra of the oligosaccharides are assumed to occupy a superposition of the spectra of each component sugar, the coupling constants of each component sugar of the oligosaccharides are almost equal to those of the corresponding monosaccharides. Based on this assumption, the best chemical shifts and coupling constants are determined by the repetition of spectral simulation using the coupling constants of the corresponding monosaccharides. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Nondestructive analysis; 1H-NMR spectrum; Oligosaccharides
1. Background Leukosialin, glycophorin A, submaxillary mucin, intestinal mucin, epitectin and fetuin, etc. are glycoproteins that have some sialyl oligosaccharides. The structure of the oligosaccharide moiety of these glycoproteins has been partially clarified, and changes in sialyl oligosaccharides with differentiation, malignant transformation and immunodeficiency have been studied extensively [1,2].
*
1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. Tel.: +81-42-778-8558; fax: +81-42-746-2144. E-mail address: [email protected] (I. Ishii-Karakasa).
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Because only a small quantity of cell surface glycans is related to these diseases, it is very important to be able to determine the complete structure of a small quantity of oligosaccharides in glycoconjugates. Various methods have recently been developed for the analysis of oligosaccharides in glycoproteins, and structural analysis with high sensitivity can now be performed in a very short time period [3]. In one such method, monosaccharides and/or oligosaccharides are analyzed with high performance liquid chromatography (HPLC), and the need for a method of structural determination of oligosaccharides by 1H-NMR spectroscopy became evident during the course of the investigation of glycoproteins in biological systems [4 –6]. However, the 1H-NMR spectra of oligosaccharides are complicated because most component sugar signals of the oligosaccharides appear in the 3.4– 4.7-ppm region [7]. While, in a structural analysis, it is very important to assign all the chemical shifts and coupling constants of the oligosaccharides (all sugar protons with the exception of OH protons), as far as we know, there are only a few reports on this subject [5,8]. In the present study, a convenient method for the analysis of oligosaccharides related to diseases was developed using HPLC and 1H-NMR spectroscopy.
2. Methods The O-linked oligosaccharides were liberated from leukosialin, glycophorin A or fetuin using endo-a-N-acetylgalactosaminidase. The reducing terminal sugar of the liberated oligosaccharides was pyridylaminated with 2-aminopyridine [9]. The pyridylamino
Fig. 1. Structure of NeuAca2-3Galb1-3 (NeuAca2-6) GalNAc-PA.
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Fig. 2. Flow chart of procedure for analysis of oligosaccharide by 1H-NMR spectrum.
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oligosaccharides were separated and purified by reversed phase HPLC. The fraction presumed to be NeuAca2-3Gal1-3 (NeuAca2-6) GalNAc-PA (I-PA; Fig. 1) with the HPLC analysis was used for the structural determination with the NMR spectrum. Prior to the NMR measurements, the pyridylamino oligosaccharide was desalted by gel filtration on a Sephadex G-10 column and lyophilized. The sample was dissolved in 0.5 ml 99.996% D2O. The 1H-NMR spectra were recorded on a Varian Unity Inova 600 spectrometer operating at 600 MHz in the Fourier transform mode. Resolution enhancement of the spectra was achieved by Lorentzian to Gaussian transformation from quadrature phase detection, followed by a complex Fourier transformation. Measurements were made at 27 C. Chemical shifts were expressed in parts per million (ppm) from the internal sodium, 4,4-dimethyl-4-silapentane-1-sulfonate (DSS), but were actually measured using internal acetone. The chemical shift of acetone compared to internal DSS was determined to be 2.225 ppm in D2O at 27 C. Since the 1H-NMR spectra of oligosaccharides are assumed to occupy a superposition of the spectra of each component sugar, the coupling constants of each component sugar of the oligosaccharides are almost equal to that of the corresponding monosaccharides. Based on this assumption, the best chemical shifts and coupling constants are determined by the repetition of spectral simulation using the coupling constants of the corresponding monosaccharides. The flow chart for the procedure is shown in Fig. 2.
3. Results GalNAc-PA, 1-methyl-Gal and 2-methyl-NeuAca were selected as the component sugars of oligosaccharide I-PA and the 1H-NMR spectra were measured. The best
Fig. 3. Resolution-enhanced 600-MHz 1H-NMR spectrum of oligosaccharide I-PA.
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chemical shifts and coupling constants of GalNAc-PA, 1-methyl-Galb and 2-methylNeuAca were obtained with the aid of LAOCOON III [10]. The 1D 1H-NMR and H – H COSY spectra of oligosaccharide I-PA were measured (Figs. 3 and 4). The chemical shifts and coupling constants of H-2, H-3, H-4, H-5 and H-60 in GalNAc that constituted oligosaccharide I-PA were easily assigned by comparing GalNAc-PA spectrum. The chemical shifts of H-1, H-10 and H-6 in GalNAc that constituted oligosaccharide IPA were assigned by the H – H COSY spectrum (see Fig. 4). However, the coupling constants of H-1, H-10 and H-6 could not be directly extracted from the spectrum because these signals appear in a region in which many signals overlap each other. Therefore, the coupling constants temporarily used were those of GalNAc-PA. Accordingly, the spectrum was calculated by the LAOCOON III program using these data.
Fig. 4. H – H COSY spectrum of oligosaccharide I-PA.
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Table 1 Best chemical shifts and coupling constants of the component GalNAc in oligosaccharide I-PA Chemical shift (d) ( ppm)
Coupling constant (Hz)
H-1 = 3.640 H-10 = 3.630 H-2 = 4.549 H-3 = 4.060 H-4 = 3.516 H-5 = 4.242 H-6 = 3.846 H-60 = 3.461
J(1, 10 ) = 13.951 J(1, 2) = 8.067 J(10, 2) = 7.684 J(2, 3) = 1.500 J(3, 4) = 9.188 J(4, 5) = 1.163 J(5, 6) = 7.653 J(5, 60 ) = 5.099 J(6, 60 ) = 10.053
Table 2 Best chemical shifts and coupling constants of the component Gal in oligosaccharide I-PA Chemical shift ( _ ) ( ppm)
Coupling constant (Hz)
H-1 = 4.590 H-2 = 3.643 H-3 = 4.108 H-4 = 3.913 H-5 = 3.630 H-6 = 3.776 H-60 = 3.733
J(1, J(2, J(3, J(4, J(5, J(5, J(6,
2) = 7.801 3) = 10.002 4) = 3.401 5) = 0.917 6) = 7.932 60 ) = 4.500 60 ) = 11.740
Table 3 Best chemical shifts and coupling constants of the component NeuAca3 in oligosaccharide I-PA Chemical shift (d) ( ppm)
Coupling constant (Hz)
H-3a = 1.815 H-3e = 2.787 H-4 = 3.670 H-5 = 3.877 H-6 = 3.675 H-7 = 3.606 H-8 = 3.882 H-9 = 3.740 H-90 = 3.547
J(3a, 3e) = 12.450 J(3a, 4) = 12.373 J(3e, 4) = 4.686 J(4, 5) = 10.396 J(5, 6) = 10.504 J(6, 7) = 1.689 J(7, 8) = 8.951 J(8, 9) = 2.625 J(8, 90 ) = 6.064 J(9, 90 ) = 11.833
a: axial; e: equatorial.
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Table 4 Best chemical shifts and coupling constants of the component NeuAca6 in oligosaccharide I-PA Chemical shift (d) ( ppm)
Coupling constant (Hz)
H-3a = 1.683 H-3e = 2.716 H-4 = 3.713 H-5 = 3.817 H-6 = 3.704 H-7 = 3.570 H-8 = 3.859 H-9 = 3.788 H-90 = 3.632
J(3a, 3e) = 12.450 J(3a, 4) = 12.283 J(3e, 4) = 4.696 J(4, 5) = 9.930 J(5, 6) = 10.165 J(6, 7) = 1.463 J(7, 8) = 9.088 J(8, 9) = 2.778 J(8, 90 ) = 6.237 J(9, 90 ) = 12.010
a: axial; e: equatorial.
The splitting pattern of H-1 that was calculated was compared with the experimental spectrum, and a similar pattern was extracted from the experimental spectrum. In this way, the experimental frequencies corresponding to the line numbers obtained in the calculation
Fig. 5. (A) Resolution-enhanced 600-MHz 1H-NMR spectrum of oligosaccharide I-PA. (B) Computer-simulated 600-MHz 1H-NMR spectrum of oligosaccharide I-PA.
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were decided. Other protons were decided in a similar manner. The spectrum of GalNAc calculated using these data was then developed by LAOCOON III. The other monosaccharides (Gal, NeuAc3 and NeuAc6 ), which constituted oligosaccharide I-PA, were analyzed in a similar manner to that already mentioned. The best chemical shifts and coupling constants of GalNAc, Gal, NeuAc3 and NeuAc6 that constituted oligosaccharide I-PA were obtained (Tables 1– 4). The structure of the oligosaccharide IPA was determined to be NeuAca2-3Galb1-3 (NeuAca2-6) GalNAc-PA (Fig. 5).
4. Discussion Cell surface glycans related to cancer and other diseases exist in very small amounts. Therefore, oligosaccharides can only be obtained in the order of micrograms after complicated separation and purification of a vast amount of the starting material. At present, the only NMR measurements possible with this quantity are 1D 1H-NMR and H – H COSY spectra. With previous methods [5], it was impossible to determine the best chemical shifts and coupling constants of I-PA because not all chemical shifts and coupling constants can be directly extracted from the 1H-NMR spectrum. We have demonstrated that our method can be applied to analyze the tetrasaccharide composed of three kinds of component sugars. With regard to 1H-NMR spectroscopy, we developed a new structural analysis to assign all the chemical shifts and coupling constants using a small quantity of oligosaccharides. This method can also be applied to the structural determination of glycans related to various other diseases. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (A) (1) (No. 09358013) from the Ministry of Education, Science, Sports and Culture of Japan. References [1] A. Varki, Biological roles of oligosaccharides, Glycobiology 3 (1993) 97 – 130. [2] A. Varki, R. Commings, J. Esko, H. Freeze, G. Hart, J. Marth, Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, New York, 1999. [3] E.F. Hounsell, Methods in Molecular Biology, Volume 76: Glycoanalysis Protocols, Humana Press, Totowa, NJ, 1998. [4] I. Ishii-Karakasa, H. Iwase, K. Hotta, Y. Tanaka, S. Omura, Partial purification and characterization of an endo-a-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242, Biochem. J. 228 (1992) 475 – 482. [5] I. Ishii-Karakasa, H. Iwase, K. Hotta, Structure determination of the O-linked sialyl oligosaccharides liberated from fetuin with endo-a-N-acetylgalactosaminidase-S by HPLC analysis and 600-MHz 1HNMR spectroscopy, Eur. J. Biochem. 247 (1997) 709 – 715. [6] I. Ishii-Karakasa, H. Iwase, K. Hotta, 1H-NMR analysis of 2-pyridylaminated O-linked sugar chains from fetuin in fetal calf serum, Bunseki Kagaku 46 (1997) 661 – 664. [7] J.P. Kamerling, J.F.G. Vliegenthart, High-resolution 1H-nuclear magnetic resonance spectroscopy of oligosaccharide-alditols related from mucin-type O-glycoproteins, Biol. Magn. Reson. 10 (1992) 1 – 287.
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[8] H. Van Halbeek, L. Dorland, G.A. Veldink, J.F.G. Vliegenthart, A 500-MHz proton-magnetic-resonance study of several fragments of the carbohydrate – protein linkage region commonly occurring in proteoglycans, Eur. J. Biochem. 127 (1982) 1 – 6. [9] S. Hase, T. Ikenaka, Y. Matsushima, Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound, Biochem. Biophys. Res. Commun. 85 (1978) 257 – 263. [10] D.F. Deter, Computer Programs for Chemistry, vol. 1, WA Benjamin, 1968, pp. 10 – 53.
A convenient method for the structural determination of glycans related to diseases Ikuko Ishii-Karakasa* Department of Biochemistry, School of Medicine, Kitasato University, Kitasato, Sagamihara, Kanagawa, Japan
Abstract A new analytical method for the structural determination of a small quantity of cell surface glycans related to diseases was developed. Oligosaccharides liberated from cell surface glycans were pyridylaminated, and pyridylamino oligosaccharides were separated and purified by high performance liquid chromatography (HPLC). The fraction presumed to be a pyridylamino tetrasaccharide on HPLC analysis was determined to be NeuAca2-3Galb1-3 (NeuAca2-6) GalNAc-PA (I-PA) by the 600-MHz 1H-NMR spectra using the following method. Since the 1HNMR spectra of the oligosaccharides are assumed to occupy a superposition of the spectra of each component sugar, the coupling constants of each component sugar of the oligosaccharides are almost equal to those of the corresponding monosaccharides. Based on this assumption, the best chemical shifts and coupling constants are determined by the repetition of spectral simulation using the coupling constants of the corresponding monosaccharides. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Nondestructive analysis; 1H-NMR spectrum; Oligosaccharides
1. Background Leukosialin, glycophorin A, submaxillary mucin, intestinal mucin, epitectin and fetuin, etc. are glycoproteins that have some sialyl oligosaccharides. The structure of the oligosaccharide moiety of these glycoproteins has been partially clarified, and changes in sialyl oligosaccharides with differentiation, malignant transformation and immunodeficiency have been studied extensively [1,2].
*
1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. Tel.: +81-42-778-8558; fax: +81-42-746-2144. E-mail address: [email protected] (I. Ishii-Karakasa).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 4 2 5 - 3
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I. Ishii-Karakasa / International Congress Series 1223 (2001) 151–159
Because only a small quantity of cell surface glycans is related to these diseases, it is very important to be able to determine the complete structure of a small quantity of oligosaccharides in glycoconjugates. Various methods have recently been developed for the analysis of oligosaccharides in glycoproteins, and structural analysis with high sensitivity can now be performed in a very short time period [3]. In one such method, monosaccharides and/or oligosaccharides are analyzed with high performance liquid chromatography (HPLC), and the need for a method of structural determination of oligosaccharides by 1H-NMR spectroscopy became evident during the course of the investigation of glycoproteins in biological systems [4 –6]. However, the 1H-NMR spectra of oligosaccharides are complicated because most component sugar signals of the oligosaccharides appear in the 3.4– 4.7-ppm region [7]. While, in a structural analysis, it is very important to assign all the chemical shifts and coupling constants of the oligosaccharides (all sugar protons with the exception of OH protons), as far as we know, there are only a few reports on this subject [5,8]. In the present study, a convenient method for the analysis of oligosaccharides related to diseases was developed using HPLC and 1H-NMR spectroscopy.
2. Methods The O-linked oligosaccharides were liberated from leukosialin, glycophorin A or fetuin using endo-a-N-acetylgalactosaminidase. The reducing terminal sugar of the liberated oligosaccharides was pyridylaminated with 2-aminopyridine [9]. The pyridylamino
Fig. 1. Structure of NeuAca2-3Galb1-3 (NeuAca2-6) GalNAc-PA.
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Fig. 2. Flow chart of procedure for analysis of oligosaccharide by 1H-NMR spectrum.
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oligosaccharides were separated and purified by reversed phase HPLC. The fraction presumed to be NeuAca2-3Gal1-3 (NeuAca2-6) GalNAc-PA (I-PA; Fig. 1) with the HPLC analysis was used for the structural determination with the NMR spectrum. Prior to the NMR measurements, the pyridylamino oligosaccharide was desalted by gel filtration on a Sephadex G-10 column and lyophilized. The sample was dissolved in 0.5 ml 99.996% D2O. The 1H-NMR spectra were recorded on a Varian Unity Inova 600 spectrometer operating at 600 MHz in the Fourier transform mode. Resolution enhancement of the spectra was achieved by Lorentzian to Gaussian transformation from quadrature phase detection, followed by a complex Fourier transformation. Measurements were made at 27 C. Chemical shifts were expressed in parts per million (ppm) from the internal sodium, 4,4-dimethyl-4-silapentane-1-sulfonate (DSS), but were actually measured using internal acetone. The chemical shift of acetone compared to internal DSS was determined to be 2.225 ppm in D2O at 27 C. Since the 1H-NMR spectra of oligosaccharides are assumed to occupy a superposition of the spectra of each component sugar, the coupling constants of each component sugar of the oligosaccharides are almost equal to that of the corresponding monosaccharides. Based on this assumption, the best chemical shifts and coupling constants are determined by the repetition of spectral simulation using the coupling constants of the corresponding monosaccharides. The flow chart for the procedure is shown in Fig. 2.
3. Results GalNAc-PA, 1-methyl-Gal and 2-methyl-NeuAca were selected as the component sugars of oligosaccharide I-PA and the 1H-NMR spectra were measured. The best
Fig. 3. Resolution-enhanced 600-MHz 1H-NMR spectrum of oligosaccharide I-PA.
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chemical shifts and coupling constants of GalNAc-PA, 1-methyl-Galb and 2-methylNeuAca were obtained with the aid of LAOCOON III [10]. The 1D 1H-NMR and H – H COSY spectra of oligosaccharide I-PA were measured (Figs. 3 and 4). The chemical shifts and coupling constants of H-2, H-3, H-4, H-5 and H-60 in GalNAc that constituted oligosaccharide I-PA were easily assigned by comparing GalNAc-PA spectrum. The chemical shifts of H-1, H-10 and H-6 in GalNAc that constituted oligosaccharide IPA were assigned by the H – H COSY spectrum (see Fig. 4). However, the coupling constants of H-1, H-10 and H-6 could not be directly extracted from the spectrum because these signals appear in a region in which many signals overlap each other. Therefore, the coupling constants temporarily used were those of GalNAc-PA. Accordingly, the spectrum was calculated by the LAOCOON III program using these data.
Fig. 4. H – H COSY spectrum of oligosaccharide I-PA.
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Table 1 Best chemical shifts and coupling constants of the component GalNAc in oligosaccharide I-PA Chemical shift (d) ( ppm)
Coupling constant (Hz)
H-1 = 3.640 H-10 = 3.630 H-2 = 4.549 H-3 = 4.060 H-4 = 3.516 H-5 = 4.242 H-6 = 3.846 H-60 = 3.461
J(1, 10 ) = 13.951 J(1, 2) = 8.067 J(10, 2) = 7.684 J(2, 3) = 1.500 J(3, 4) = 9.188 J(4, 5) = 1.163 J(5, 6) = 7.653 J(5, 60 ) = 5.099 J(6, 60 ) = 10.053
Table 2 Best chemical shifts and coupling constants of the component Gal in oligosaccharide I-PA Chemical shift ( _ ) ( ppm)
Coupling constant (Hz)
H-1 = 4.590 H-2 = 3.643 H-3 = 4.108 H-4 = 3.913 H-5 = 3.630 H-6 = 3.776 H-60 = 3.733
J(1, J(2, J(3, J(4, J(5, J(5, J(6,
2) = 7.801 3) = 10.002 4) = 3.401 5) = 0.917 6) = 7.932 60 ) = 4.500 60 ) = 11.740
Table 3 Best chemical shifts and coupling constants of the component NeuAca3 in oligosaccharide I-PA Chemical shift (d) ( ppm)
Coupling constant (Hz)
H-3a = 1.815 H-3e = 2.787 H-4 = 3.670 H-5 = 3.877 H-6 = 3.675 H-7 = 3.606 H-8 = 3.882 H-9 = 3.740 H-90 = 3.547
J(3a, 3e) = 12.450 J(3a, 4) = 12.373 J(3e, 4) = 4.686 J(4, 5) = 10.396 J(5, 6) = 10.504 J(6, 7) = 1.689 J(7, 8) = 8.951 J(8, 9) = 2.625 J(8, 90 ) = 6.064 J(9, 90 ) = 11.833
a: axial; e: equatorial.
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Table 4 Best chemical shifts and coupling constants of the component NeuAca6 in oligosaccharide I-PA Chemical shift (d) ( ppm)
Coupling constant (Hz)
H-3a = 1.683 H-3e = 2.716 H-4 = 3.713 H-5 = 3.817 H-6 = 3.704 H-7 = 3.570 H-8 = 3.859 H-9 = 3.788 H-90 = 3.632
J(3a, 3e) = 12.450 J(3a, 4) = 12.283 J(3e, 4) = 4.696 J(4, 5) = 9.930 J(5, 6) = 10.165 J(6, 7) = 1.463 J(7, 8) = 9.088 J(8, 9) = 2.778 J(8, 90 ) = 6.237 J(9, 90 ) = 12.010
a: axial; e: equatorial.
The splitting pattern of H-1 that was calculated was compared with the experimental spectrum, and a similar pattern was extracted from the experimental spectrum. In this way, the experimental frequencies corresponding to the line numbers obtained in the calculation
Fig. 5. (A) Resolution-enhanced 600-MHz 1H-NMR spectrum of oligosaccharide I-PA. (B) Computer-simulated 600-MHz 1H-NMR spectrum of oligosaccharide I-PA.
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were decided. Other protons were decided in a similar manner. The spectrum of GalNAc calculated using these data was then developed by LAOCOON III. The other monosaccharides (Gal, NeuAc3 and NeuAc6 ), which constituted oligosaccharide I-PA, were analyzed in a similar manner to that already mentioned. The best chemical shifts and coupling constants of GalNAc, Gal, NeuAc3 and NeuAc6 that constituted oligosaccharide I-PA were obtained (Tables 1– 4). The structure of the oligosaccharide IPA was determined to be NeuAca2-3Galb1-3 (NeuAca2-6) GalNAc-PA (Fig. 5).
4. Discussion Cell surface glycans related to cancer and other diseases exist in very small amounts. Therefore, oligosaccharides can only be obtained in the order of micrograms after complicated separation and purification of a vast amount of the starting material. At present, the only NMR measurements possible with this quantity are 1D 1H-NMR and H – H COSY spectra. With previous methods [5], it was impossible to determine the best chemical shifts and coupling constants of I-PA because not all chemical shifts and coupling constants can be directly extracted from the 1H-NMR spectrum. We have demonstrated that our method can be applied to analyze the tetrasaccharide composed of three kinds of component sugars. With regard to 1H-NMR spectroscopy, we developed a new structural analysis to assign all the chemical shifts and coupling constants using a small quantity of oligosaccharides. This method can also be applied to the structural determination of glycans related to various other diseases. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (A) (1) (No. 09358013) from the Ministry of Education, Science, Sports and Culture of Japan. References [1] A. Varki, Biological roles of oligosaccharides, Glycobiology 3 (1993) 97 – 130. [2] A. Varki, R. Commings, J. Esko, H. Freeze, G. Hart, J. Marth, Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, New York, 1999. [3] E.F. Hounsell, Methods in Molecular Biology, Volume 76: Glycoanalysis Protocols, Humana Press, Totowa, NJ, 1998. [4] I. Ishii-Karakasa, H. Iwase, K. Hotta, Y. Tanaka, S. Omura, Partial purification and characterization of an endo-a-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242, Biochem. J. 228 (1992) 475 – 482. [5] I. Ishii-Karakasa, H. Iwase, K. Hotta, Structure determination of the O-linked sialyl oligosaccharides liberated from fetuin with endo-a-N-acetylgalactosaminidase-S by HPLC analysis and 600-MHz 1HNMR spectroscopy, Eur. J. Biochem. 247 (1997) 709 – 715. [6] I. Ishii-Karakasa, H. Iwase, K. Hotta, 1H-NMR analysis of 2-pyridylaminated O-linked sugar chains from fetuin in fetal calf serum, Bunseki Kagaku 46 (1997) 661 – 664. [7] J.P. Kamerling, J.F.G. Vliegenthart, High-resolution 1H-nuclear magnetic resonance spectroscopy of oligosaccharide-alditols related from mucin-type O-glycoproteins, Biol. Magn. Reson. 10 (1992) 1 – 287.
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[8] H. Van Halbeek, L. Dorland, G.A. Veldink, J.F.G. Vliegenthart, A 500-MHz proton-magnetic-resonance study of several fragments of the carbohydrate – protein linkage region commonly occurring in proteoglycans, Eur. J. Biochem. 127 (1982) 1 – 6. [9] S. Hase, T. Ikenaka, Y. Matsushima, Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound, Biochem. Biophys. Res. Commun. 85 (1978) 257 – 263. [10] D.F. Deter, Computer Programs for Chemistry, vol. 1, WA Benjamin, 1968, pp. 10 – 53.