RESUMO
The kinetic parameters of the first and second oxygenation of arachidonic acid by soybean lipoxygenase-1 were determined and found to be for the first step at pH 10.0, Km (arachidonic acid) = 8.5 +/- 0.5 microM; kcat = 225 +/- 7 s-1 and for the second step at pH 8.7 Km (15-HPETE) = 440 +/- microM; kcat = 25 +/- 1 s-1. In the second oxygenation for which 15-Ls-hydroperoxy 5-cis, 8-cis, 11-cis 13-trans-eicosatetraenoic acid is a substrate, two isomeric dihydroperoxy fatty acids are formed. After separation of the corresponding dihydroxy esters by high-performance liquid chromatography, they were identified by mass-spectrometry, 1H- and 13C-NMR spectroscopy as 8-DS, 15-LS-dihydroperoxy 5-cis, 9-trans, 11-cis, 13-trans-eicosatetraenoic acid and 5-DS, 15-LS-dihydroperoxy 6-trans, 8-cis, 11-cis, 13-trans-eicosatetraenoic acid. Independent evidence for the absolute configurations was obtained by capillary gas-liquid chromatography of diastereomeric R-(-)-2-butyl esters of the acetylated 2-hydroxy carboxylic acids produced by oxidative ozonolysis of the acetylated dihydroxy fatty acids. It is concluded that soybean lipoxygenase-1 produces hydroperoxides with predominantly the S-configuration irrespective of the position in the fatty acid which is oxygenated.
Assuntos
Ácidos Araquidônicos , Lipoxigenase/metabolismo , Plantas/enzimologia , Araquidonato Lipoxigenases , Ácidos Araquidônicos/síntese química , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Métodos , Glycine max , Espectrofotometria Ultravioleta , Especificidade por SubstratoRESUMO
The elucidation of the structures of two carbohydrate units, N-glycosidically linked to an asparagine residue of bovine lactotransferrin, is described. These carbohydrate structures are of the oligomannoside type and contain eight or nine mannose residues, respectively. The potency of 500 MHz 1H-NMR spectroscopy in primary structure determination of two closely related carbohydrate chains present in a mixture is demonstrated. This implies that 500 MHz 1H-NMR spectroscopy can disclose microheterogeneity which is almost untraceable using other approaches.
Assuntos
Lactoferrina , Lactoglobulinas , Animais , Configuração de Carboidratos , Bovinos , Espectroscopia de Ressonância MagnéticaRESUMO
360-MHz 1H-NMR spectra were recorded of NeuAc alpha(2 leads to 3)Gal beta (1 leads to 3)GalNAc-ol (I), Gal beta(1 leads to 3)[NeuAc alpha(2 leads to 6)] GalNAc-ol (II) and NeuAc alpha (2 leads to 3)-Gal beta(1 leads to 3) [NeuAc alpha(2 leads to 6)]GalNAc-ol (III). The chemical shifts and coupling constants of the anomeric protons, the H-3ax and H-3eq of NeuAc, the GalNAc-ol skeleton protons, the H-3 of Gal and the N-acetyl protons of GalNAc-ol and NeuAc provide conclusive evidence for the identification of the primary structures. Compound II represents a novel carbohydrate chain of kappa-casein.
Assuntos
Caseínas , Oligossacarídeos/análise , Sequência de Aminoácidos , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Bovinos , Espectroscopia de Ressonância Magnética , Ácidos Siálicos/análiseRESUMO
The lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum were extracted from cytoplasmic membranes or from disintegrated bacteria with aqueous phenol and purified by gel chromatography. The lipoteichoic acid preparations contained phosphate, glycerol, galactose, glucose and fatty acids in a molar ratio of 1.0:1.0:1.3:1.2:0.3. Chemical analysis and NMR studies of the native preparations and of products from various acid and alkaline hydrolysis procedures gave evidence for the structure of two lipoteichoic acids. The lipid anchor appeared to be 3-O-(6'-(sn-glycero-1-phosphoryl)diacyl-beta-D-galactofuranosyl)-sn-1, 2-diacylglycerol. The polar part showed two structural features not previously described for lipoteichoic acids. A 1,2-(instead of the usual 1,3-) phosphodiester-linked sn-glycerol phosphate chain is only used substituted at the terminal glycerol unit with a linear polysaccharide, containing either beta(1----5)-linked D-galactofuranosyl groups or beta(1----6)-linked D-glucopyranosyl groups.
Assuntos
Bifidobacterium/análise , Lipopolissacarídeos , Ácidos Fosfatídicos/isolamento & purificação , Ácidos Teicoicos/isolamento & purificação , Carboidratos/análise , Membrana Celular/análise , Hidrólise , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , MetilaçãoRESUMO
Acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz 1H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man5GlcNAcGlcNAc-ol and Man7GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man2-3GlcNAc[Fuc]0-1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.
Assuntos
Glicoproteínas , alfa-Glucosidases , Sequência de Carboidratos , Glicoproteínas/análise , Humanos , Espectroscopia de Ressonância Magnética , Peso Molecular , Placenta/enzimologia , alfa-Glucosidases/análiseRESUMO
The structure of the carbohydrate chains of mucous glycoproteins from the gastro-intestinal tract was examined for species- and tissue-specificity. To this purpose, oligosaccharides were released from purified glycoprotein preparations of rat and pig gastric, duodenal-gland and small-intestinal mucus, by alkaline borohydride reductive cleavage. Based on the results of 500-MHz 1H-NMR spectroscopy and of sugar analysis of the total oligosaccharide fractions, terminal GlcNAc, alpha (1 leads to 4)-linked to galactose, appears to be a characteristic constituent of duodenal-gland oligosaccharides. Similarly, NeuAc in alpha (2 leads to 3)-linkage to galactose turns out to be a typical constituent of small-intestinal mucous glycoproteins. In general, glycoproteins from gastric mucus possess larger and more-branched carbohydrate chains than those from duodenal-gland and small-intestinal mucus. Comparing rat and pig, oligosaccharide structures for corresponding tissues are less complex for the former. After fractionation, the rat duodenal-gland oligosaccharides could be characterized by application of 1H-NMR spectroscopy as being branched tetra- up to hexa-saccharide chains, all sharing the italicized trisaccharide element. The chains exhibit microheterogeneity as to the termination by fucose in alpha (1 leads to 2)- or by GlcNAc in alpha (1 leads to 4)-linkage to galactose. The following structures can be proposed for the most abundant rat duodenal-gland oligosaccharides: (table; see text).
Assuntos
Acetilglucosamina/análise , Duodeno/análise , Mucosa Gástrica/análise , Glucosamina/análogos & derivados , Glicoproteínas , Mucosa Intestinal/análise , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Glicoproteínas/isolamento & purificação , Espectroscopia de Ressonância Magnética , Masculino , Oligossacarídeos/análise , Especificidade de Órgãos , Ratos , Ratos Endogâmicos , Especificidade da Espécie , Relação Estrutura-Atividade , SuínosRESUMO
The oligosaccharides of chick embryo type I procollagen were isolated from carboxyl-terminal propeptide fragment by exhaustive digestion with papain and pronase, and then purified as a mixture of glycopeptides. The structures of the oligosaccharides were established by high-resolution 1H-NMR spectroscopy and found to be a mixture with respect to the non-reducing terminal residues as shown below: (formula; see text) The percentages refer to the relative amount of those mannose residues present in the mixture. The data suggest that the oligosaccharides are a microheterogeneous mixture of high-mannose type glycans containing between six and nine mannose residues per carbohydrate unit. Such carbohydrate chains, although not uncommon for glycoproteins, had never been found before for collagen or collagen-related compounds.
Assuntos
Pró-Colágeno/análise , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Embrião de Galinha , Espectroscopia de Ressonância Magnética , Oligossacarídeos/análise , Papaína/metabolismo , Pronase/metabolismoRESUMO
During our studies of bovine brain neutral glycosphingolipids (Ngsl's), we have purified a compound that co-migrates on thin-layer chromatogram with standard GA1 (purified by acid hydrolysis of GM1) and close to penta- (nLc5Cer) glycosylceramide from bovien erythrocytes. The structure of the purified Ngsl from brain has been established by permethylation and by stepwise exoglycosidase hydrolysis. 600 MHz 1H NMR spectroscopy of the oligosaccharide obtained from the Ngsl after endoglycoceramidase hydrolysis confirms the structure as ganglio-N-tetraosylceramide (GgOse4Cer or GA1) as Gal beta 1----3GalNAc beta 1----4Gal beta 1----4Glc beta 1----1Cer. We have identified GA1 in bovine, rat and human brain and myelin by TLC-immunostaining with monospecific anti-GA1 antiserum.
Assuntos
Química Encefálica , Gangliosídeo G(M1)/análise , Bainha de Mielina/química , Animais , Sequência de Carboidratos , Bovinos , Cromatografia em Camada Fina , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência MolecularRESUMO
Alkaline borohydride reductive cleavage (beta-elimination) of desialylated human kappa-caseinoglycopeptide resulted in the release of a series of oligosaccharides. The smaller-size compounds among them were purified to virtual homogeneity by gel filtration followed by high-performance liquid chromatography. The structures of 9 oligosaccharides were determined by 1H-NMR spectroscopy in conjunction with sugar analysis. The tetrasaccharide Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol and various partial structures thereof were characterized. Notably, the disaccharide GlcNAc beta(1----6)GalNAc-ol and the trisaccharide Gal beta(1----4)GlcNAc beta(1----6)GalNAc-ol were identified; they represent a novel type of core structure for mucin-type carbohydrate chains, namely a peptide-linked GalNAc that is mono-substituted at C-6. In addition, some oligosaccharides ending in GlcNAc-ol could be characterized. Their possible origin is discussed.
Assuntos
Caseínas/análise , Mucinas/análise , Oligossacarídeos/isolamento & purificação , Configuração de Carboidratos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Humanos , Espectroscopia de Ressonância Magnética , Leite Humano/análiseRESUMO
Glycopeptides obtained from human serotransferrin by pronase digestion were separated into two fractions by affinity chromatography on Con A-Sepharose. The retarded fraction (85% of total glycopeptides) contained sialylated biantennary glycans of the N-acetyllactosaminic type, the primary structure of which has been previously determined. The non-retained fraction (15% of total glycopeptides) consisted of two isomeric triantennary glycans of the N-acetyllactosaminic type. The primary structure have been elucidated by methylation analysis and 500 MHz 1H-NMR spectroscopy. Both contain an additional NeuAc(alpha 2----3)Gal(beta 1----4)GlcNAc antenna. The latter is linked to C-4 of the (alpha 1----3) bound Man residue in 45% of the glycans in the non-retained fraction but to C-6 of the (alpha 1----6) bound Man residue, in the remaining 55% of the glycans in this fraction.
Assuntos
Polissacarídeos , Transferrina , Sequência de Carboidratos , Glicopeptídeos/isolamento & purificação , Glicopeptídeos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Pronase/metabolismoRESUMO
N-glycosidically-linked glycans released by hydrazinolysis of human factor VIII/von Willebrand factor (FVIII/vWf) were separated by high-voltage electrophoresis. Five fractions were obtained, one of them representing 60% of the total amount of the N-glycosidically-linked glycans of FVIII/vWf. On the basis of the carbohydrate composition, methylation analysis and 500 MHz 1H-NMR spectroscopy, we describe the primary structure of this major glycan which is of the monosialylated and monofucosylated biantennary N-acetyllactosaminic type.
Assuntos
Asparagina , Fatores de Coagulação Sanguínea , Carboidratos , Fator VIII , Fator de von Willebrand , Fatores de Coagulação Sanguínea/análise , Configuração de Carboidratos , Sequência de Carboidratos , Carboidratos/análise , Fator VIII/análise , Humanos , Hidrazinas , Espectroscopia de Ressonância Magnética , Metilação , Oligossacarídeos/análise , Fator de von Willebrand/análiseRESUMO
The carbohydrate chains of the pathological human immunoglobulins M from two patients with Waldenström's macroglobulinemia were released by hydrazinolysis. The N-acetyllactosamine-type glycans were obtained by affinity chromatography on concanavalin A and fractionated by high-voltage paper electrophoresis. The primary structure of the major compounds was elucidated on the basis of carbohydrate analysis, methylation analysis, including mass-spectrometry, and 500 MHz 1H-NMR spectroscopy. For both patients, this appeared to be a monosialyl monofucosyl biantennary structure; the compounds differed by the presence of an intersecting N-acetylglucosamine residue.
Assuntos
Amino Açúcares/isolamento & purificação , Imunoglobulina M/análise , Polissacarídeos/isolamento & purificação , Macroglobulinemia de Waldenstrom/imunologia , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia de Afinidade , Eletroforese em Papel , Humanos , Espectroscopia de Ressonância Magnética , MetilaçãoRESUMO
Glycocalicin (140 kDa), the main constituent of the glycoprotein Ib alpha-chain (150 kDa) of the human platelet membrane, contains 4 putative N-glycosylation sites. For the structural analysis of the N-glycosidic carbohydrate chains of glycocalicin, the glycoprotein has been subjected to the hydrazinolysis procedure. The acidic carbohydrate chains obtained were fractionated by ion-exchange chromatography on DEAE-Sephadex A-25, and subsequently analyzed by sugar analysis, anion-exchange chromatography on Mono Q HR 5/5 and 500 MHz 1H-NMR spectroscopy. A novel tetrasialylated monofucosylated tetraantennary chain was identified in the glycoprotein. It could also be deduced that in all structures the alpha 2----6-linked NeuAc is attached exclusively at the Gal beta 1----2Man alpha 1----3 antenna, whereas the other antennae can be terminated with alpha 2----3-linked NeuAc. As minor constituents sialylated N-linked carbohydrate chains with a terminal Fuc alpha 1----2Gal beta 1----sequence were detected.
Assuntos
Plaquetas/análise , Carboidratos/análise , Fucose/análise , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteínas da Membrana de Plaquetas/análise , Ácidos Siálicos/análise , Amino Açúcares/análise , Configuração de Carboidratos , Membrana Celular/análise , Cromatografia por Troca Iônica , Glicosilação , Humanos , Hidrazinas , Espectroscopia de Ressonância Magnética , Ácido N-AcetilneuramínicoRESUMO
The N-glycosidic carbohydrate chains of hen beta-ovomucoid were released from the protein by hydrazinolysis, and separated by HPLC. Primary structural analysis of 3 major fractions was conducted by applying 500-MHz 1H-NMR spectroscopy in combination with methylation analysis. One of the fractions investigated appeared to consist of an intersected penta-antennary structure extended with one Gal residue. The location of the latter in a certain branch could be established unambiguously by NMR. This structure is a novel member of the family of N-glycosidic carbohydrates of glycoproteins.
Assuntos
Proteínas do Ovo , Ovomucina , Animais , Fenômenos Químicos , Química , Físico-Química , Galinhas , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Glicosídeos/análise , Espectroscopia de Ressonância Magnética , MetilaçãoRESUMO
After exhaustive pronase digestion, purification by gel filtration and affinity chromatography on concanavalin A, three glycopeptide fractions were obtained from rat hemopexin. Two fractions (I and II) were concanavalin A non-reactive and one (III) was concanavalin A reactive. On the basis of carbohydrate composition, methylation analysis and proton nuclear magnetic resonance spectroscopy, the primary structure of the glycan in fraction III is proposed as being a mixture of mono- and di-sialo-diantennae of the N-glycosidic, N- acetyllactosamine type. Hydrazinolysis of glycopeptides not binding to concanavalin A yielded mixtures of oligosaccharides for both fractions. These oligosaccharides were separated by HPLC; the molar composition of each of them is given. These data suggest that rat hemopexin contains, among others, a diantennary structure bearing three sialic acid residues.
Assuntos
Hemopexina/análise , Polissacarídeos/análise , Animais , Carboidratos/análise , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Pronase/metabolismo , RatosRESUMO
The procedure for the purification of the adhesin polysaccharide receptor and its hexasaccharide repeating unit from whole S. oralis ATCC 55229 by chemical, enzymatic, and chromatographic techniques has been described. Chemical, chromatographic, and mass spectrometric procedures allow preliminary structural characterization of the hexasaccharide repeating unit and polysaccharide. The structural characterizations of the hexasaccharide and polysaccharide are completed using several 1D and 2D NMR techniques. Identification of the anomeric 1H and 13C signals of the glycosyl residues permits, by virtue of their chemical shifts and coupling constants (3JHH and 1JCH), the determination of the configurations of the glycosidic linkages. The HMBC connectivities permit the establishment of the hexasaccharide sequence as Rhap alpha(1-->2)Rhap alpha(1-->3)Galp alpha(1-->3)Galp beta(1-->4)Glcp beta(1-->3)Gal. The 1H NMR chemical shifts of the polysaccharide, as determined by the combination of COSY and TOCSY experiments, and the observed interglycosidic NOESY cross-peaks reveal the structure of the polysaccharide to be [formula: see text] where the position of the glycerol (Gro) phosphate moiety has been determined by [1H, 31P] NMR spectroscopy.
Assuntos
Adesinas Bacterianas/metabolismo , Polissacarídeos Bacterianos/isolamento & purificação , Receptores Imunológicos/isolamento & purificação , Sequência de Carboidratos , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Polissacarídeos Bacterianos/química , Receptores Imunológicos/química , Streptococcus/química , Streptococcus sanguis/químicaRESUMO
Lysosomal acid alpha-mannosidase from porcine kidney was found to contain mannose (4.8%), galactose (0.9%), fucose (0.5%), N-acetylglucosamine (3.1%), and mannose 6-phosphate (0.1%). Approximately 50% of the total hexose of the oligosaccharide chains could be released by endo-beta-N-acetylglucosaminidase-H (endo-H). They were predominantly neutral, oligomannoside-type oligosaccharides containing 5, 6, and 9 mannose residues, respectively, in the centesimal ratio of 36:25:34. 500-MHz 1H-NMR spectroscopy in conjunction with sequential exoglycosidase digestion of the reduced compounds revealed that each of the three fractions consisted of a single isomer only; the Man9 compound has the following structure: (Formula: see tex). The Man6-compound lacks Man residues D1, D2, and D3, while the Man5-compound lacks Man-C as well. In addition to the neutral ones, some (5%) phosphorylated oligomannoside-type oligosaccharides were obtained. The endo-H resistant glycopeptides were subjected to hydrazinolysis. Approximately 60% of the oligosaccharides released by hydrazine were found to be of rather small size; their composition can be represented asMan2-3GlcNAc[Fuc]0-1GlcNAcol. The remaining 40% consist of larger-size galactose-containing, N-acetyllactosamine-type oligosaccharides. Studies involving sequential exoglycosidase digestion and 500-MHz 1H-NMR spectroscopy performed on the highly purified small-sized compounds revealed the following four structures for the endo-H-resistant oligosaccharides: (Formula: see text).
Assuntos
Rim/enzimologia , Manosidases/isolamento & purificação , Oligossacarídeos/análise , Animais , Carboidratos/análise , Cromatografia em Gel/métodos , Eletroforese em Gel de Poliacrilamida , Hidrazinas/farmacologia , Hidrogênio , Espectroscopia de Ressonância Magnética , Oligossacarídeos/isolamento & purificação , Análise Espectral/métodos , Suínos , alfa-ManosidaseRESUMO
The carbohydrate portions of beta-galactosidase from Aspergillus oryzae were found to be composed of two types of sugar chains. They were released equally well with endo-beta-N-acetylglucosaminidase H, but were distinct in their chain length. The long sugar chains (fraction I), corresponding to 4% of the total carbohydrate chains, were composed of galactomannan-type oligosaccharides, which consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 30.0, 16.4, 1.4, and 2.1 per mol of aspartic acid, respectively. The short sugar chains (fraction II), corresponding to 96% of the total carbohydrate chains, consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 9.4, 0.6, 0.3, and 1.7 per mol of aspartic acid, respectively. Both types of sugar chains were fractionated into neutral and acidic subfractions. The neutral subfraction of fraction I (I-N), corresponding to 1% of the total carbohydrate chains, was very heterogeneous in length and was resistant to digestion with alpha-mannosidase and beta-galactosidase. The neutral subfraction of fraction II (II-N), corresponding to 91% of the total carbohydrate, was composed of a mixture of oligosaccharides with oligomanneoside chains (Mann GlcNAcol). The major components were similar to high mannose-type oligosaccharides of mammalian origin in their composition and size (n = 5-9). However, digestion of II-N with alpha 1,2-mannosidase produced considerable amounts of Man6GlcNAcol, an unusual product in the case of high mannose-type oligosaccharides of mammalian origin, in addition to the common one, Man5GlcNAcol.
Assuntos
Aspergillus oryzae/enzimologia , Aspergillus/enzimologia , Galactosidases/isolamento & purificação , Oligossacarídeos/isolamento & purificação , beta-Galactosidase/isolamento & purificação , Aminoácidos/análise , Sequência de Carboidratos , Glicopeptídeos/isolamento & purificação , Dados de Sequência MolecularRESUMO
We propose a phase-sensitive gradient-enhanced two-dimensional heteronuclear multiple-bond correlation (psge-2D HMBC) experiment for speedy, accurate, and precise measurement of 2JCH and 3JCH. The experiment does not suppress one-bond correlations. Rather, the value of a desired long-range JCH is obtained from the pertinent cross-peak pattern in the HMBC spectrum, using the corresponding 1JCH correlation pattern as reference. The application of the proposed experiment is illustrated for the trisaccharide raffinose.
Assuntos
Processamento de Imagem Assistida por Computador , Espectroscopia de Ressonância Magnética/métodos , Rafinose/química , Análise de Fourier , Estrutura Molecular , Reprodutibilidade dos Testes , SoftwareRESUMO
A 21-amino acid glycopeptide (Gp21) was isolated and purified in multi-milligram yields from commercially available human serum transferrin (HSTF) by a combination of tryptic digestion, Con A affinity chromatography, and reverse phase HPLC. The peptide chain of Gp21 contains a single N-glycosylation site to which a diantennary oligosaccharide is attached. The amino acid sequence and the glycan primary structure of Gp21 have been verified by peptide sequencing, electrospray mass spectrometry, and one-dimensional 1H NMR spectroscopy. Different glycoforms were found for the glycan of Gp21 derived from two different batches of commercial HSTF. These glycoforms differ from one another in the number of NeuAc residues (ranging from 0 to 2) and/or the number of Gal residues (ranging from 1 to 2). As for the monogalacto species, in the two-dimensional nuclear Overhauser effect (NOE) spectrum of Gp21, interglycosidic NOEs were observed between Man4 in the alpha (1-->3) branch and the terminal GlcNAc beta (1-->2) residue. No interglycosidic NOE was observed between Man4' in the alpha (1-->6) branch and the terminal GlcNAc residue. These observations indicate that the terminal GlcNAc residue in the minor glycoforms of Gp21 is exclusively located in the alpha (1-->3) branch of the Gp21 glycan. The occurrence of such a carbohydrate structure in HSTF has not been reported before. The 1H and 13C NMR spectra of Gp21 have been completely assigned by two-dimensional homonuclear and heteronuclear spectroscopy. The close similarity of the 1H and 13C chemical shift values for the Gp21 glycan with the respective values for the peptide-free diantennary oligosaccharide (Wieruszeski et al., Glycoconjugate J., 6 (1989) 183-194) indicates that the 1H and 13C chemical shifts of the diantennary oligosaccharide are not perturbed by the presence of the Gp21 peptide fragment. The complete 1H and 13C resonance assignments and the full characterization of the primary structure of Gp21 will permit us to study the conformation and dynamics of the N-linked diantennary oligosaccharides while covalently attached to a polypeptide fragment.