An improved procedure for the methylation analysis of oligosaccharides and polysaccharides

Harris, Philip J.; Henry, Robert J.; Blakeney, A. B.; Stone, Bruce

doi:10.1016/0008-6215(84)85106-x

Cited by 565 publications

(227 citation statements)

References 31 publications

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“…The loss of the Rib during linkage analysis is not unexpected if the Rib was a constituent of charged molecules. During the methylation procedure used for linkage analysis (Harris et al, 1984), permethylated polysaccharides were extracted from the methylation mixture by partitioning between water and chloroform. Neutral permethylated polysaccharides are readily extracted into the organic solvent phase, whereas the salts and ions partition into the aqueous phase, which is discarded.…”

Section: Discussionmentioning

confidence: 99%

“…Constituent monosaccharides were converted to their corresponding alditol acetates by trifluoroacetic acid-hydrolysis, reduction with NaB 2 H 4 , and acetylation essentially following the protocol described by Harris et al (1984). The alditol acetates, including glycerol triacetate, were separated, identified, and quantified using GC-MS as described previously (Chiovitti et al, 2003).…”

Section: Carbohydrate Analysesmentioning

confidence: 99%

“…The alditol acetates, including glycerol triacetate, were separated, identified, and quantified using GC-MS as described previously (Chiovitti et al, 2003). To investigate the linkage patterns of the constituent monosaccharides, samples were subjected to methylation analysis as described by Harris et al (1984), except that NaOH in (CH 3 ) 2 SO was used to generate the alkoxide (Ciucanu & Kerek, 1984). The permethylated alditol acetates were separated and analysed by the same methods described for constituent monosaccharide analysis (Chiovitti et al, 2003).…”

Section: Carbohydrate Analysesmentioning

confidence: 99%

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The glucans extracted with warm water from diatoms are mainly derived from intracellular chrysolaminaran and not extracellular polysaccharides

Chiovitti

Molino

Crawford

et al. 2004

European Journal of Phycology

100

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Several recent studies have employed warm-water treatment of diatom cells to extract nominally bound extracellular polymeric substances. Where examined, the dominant neutral sugar in these extracts was glucose. In the present study, we sought to characterize the structure of the glucose-rich polymers in the water extracts of diatoms and to determine the origin of these polymers. The marine diatoms surveyed were Phaeodactylum tricornutum, Cylindrotheca fusiformis, Craspedostauros australis and Thalassiosira pseudonana. A freshwater species, Pinnularia viridis, was also investigated for the dye labelling experiments. Freshly harvested marine diatoms were extracted with water at 308C for 1 h. Constituent monosaccharide analyses showed that glucose was the dominant neutral sugar (80 -95 mol% of the total) in the extracts from three marine species, whereas the P. tricornutum extract contained predominantly ribose, galactose and glucose, and was inferred to be enriched in low-molecular-weight components. Linkage analysis of the constituent monosaccharides and proton nuclear magnetic resonance spectroscopy showed that the glucose in these extracts was derived primarily from 1,3-b-D-glucan. Immunocytochemistry with a monoclonal anti-1,3-b-D-glucan antibody confirmed that the glucan was localized in the vacuoles of diatom cells preserved by freeze-substitution. Nearly all diatom cells incubated with a fluorescent dye, DiBAC 4 (3), during warm water treatment at 308C or 608C incorporated the dye, demonstrating that the membrane integrity of the diatoms was compromised and supporting the contention that intracellular glucan was released during the treatment. In light of these data, the extracellular glucans of diatoms reported in some previous studies are re-interpreted as intracellular chrysolaminaran.

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Section: Discussionmentioning

confidence: 99%

Section: Carbohydrate Analysesmentioning

confidence: 99%

Section: Carbohydrate Analysesmentioning

confidence: 99%

See 1 more Smart Citation

The glucans extracted with warm water from diatoms are mainly derived from intracellular chrysolaminaran and not extracellular polysaccharides

Chiovitti

Molino

Crawford

et al. 2004

European Journal of Phycology

100

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“…Glycosidic-linkage composition was determined by gas chromatography-quadrupole mass spectrometry (GC-qMS) of the partially methylated alditol acetates [25][26][27]. Briefly, the polysaccharides were methylated with 80 lL of methyl iodide and the mixture was allowed to react for 20 min under stirring.…”

Section: Methylation Analysismentioning

confidence: 99%

Improved efficiency of brewer’s spent grain arabinoxylans by ultrasound-assisted extraction

Reis¹,

Coelho

Coimbra

et al. 2015

Ultrasonics Sonochemistry

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“…The fully methylated products were purified according to [17] using acidified 2,2-dimethoxypropane. The resulting purified products were hydrolysed under different acid concentrations including the acetolysis procedure described by Stellner et al [18].…”

Section: Methylation Analysismentioning

confidence: 99%

Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029

Basu

Kuhn

Neszmélyi

et al. 1987

European Journal of Biochemistry

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Serologically characterized samples of enterobacterial common antigen (ECA) from Plesiomonas shigelloides, Salmonella montevideo and Shigella sonnei were investigated by chemical methods including methylation and NMR techniques. All showed the same sugar composition and contained a lipid moiety with palmitic acid as main fatty acid and with a phosphodiester group. Additional enzymatic studies, reported in the preceding paper, provided evidence that the lipid moiety is an L-glycerophosphatidyl residue attached via a phosphodiester linkage to C-1 of GlcNAc as the reducing end of the ECA sugar chain.ECA of P. shigelloides showed the best-resolved "C-NMR spectra, especially after the removal of nonstoichiometric O-acetyl groups at C-6 of GlcNAc of the ECA repeating unit and of the lipid moiety by mild acid hydrolysis (0.01 M HC1, 100"C, 10 min). Subsequent I3C-NMR studies were therefore carried out with the mildacid-treated ECA of P. shigelloides which allowed a tentative assignment of all resonances of the ECA repeating unit. 13C-NMR spectra of Salmonella and Shigella ECA were essentially the same as those obtained with Plesiomonas ECA.The same trisaccharide repeating unit was encountered as demonstrated previously in the cyclic form of ECA isolated from S. sonnei by Dell et al. [Carbohydr. Res. 133, 95-104 (1984)l. Methylation analysis, however, afforded small amounts of terminal GlcNAc thus proving, in combination with the demonstration of the attached lipid moiety, an acyclic nature of ECA from P. shigelloides and from the two enterobacterial species. The question of whether the cyclic form co-exists in S. sonnei phase I and possibly in other enterobacterial species or, whether it had been formed during extraction as an artifact, has not yet been answered. The way in which ECA was isolated in our studies would preclude the presence of a non-amphiphilic (cyclic) polysaccharide.The finding that the sugar chain of ECA is attached to an L-glycerophosphatidyl residue is in full corroboration with serological, enzymatical and gel electrophoretic studies shown in the preceding paper and with the character of ECA as a surface antigen being anchored by hydrophobic interactions in the outer membrane of Enterobacteriaceae and P. shigelloides. Abbreviations. ECA, enterobacterial common antigen; p, pyranose; GlcpNAc, 2-acetamido-2-deoxy-~-glucopyranose; ManpNAc, 2-acetamido-2-deoxy-~-mannopyranose; ManpNAcA, 2-acetamido-2-deoxy-~-mannopyranosyl uronic acid; Fucp4NAc, 4-acetamido-4-deoxy-D-fucopyranose; Me, methyl; Ac, acetyl; GLC, gas-liquid chromatography; GLC-MS, combined gas-liquid chromatography/mass spectrometry; ELISA, enzyme-linked immunosorption assay; FAB-MS, fast atom bombardment/mass spectrometry; El, electron impact mass spectrometry.[3, 61. With these mutants immunogenic and haptenic ECA can be separately obtained using the extraction method of Mannel and Mayer [7]. A cyclic form of ECA was recently described by Dell et al. [8] from Shigella sonnei phase I cells extracted by the technique of Lugowski et al. [9]...

show abstract

An improved procedure for the methylation analysis of oligosaccharides and polysaccharides

Cited by 565 publications

References 31 publications

The glucans extracted with warm water from diatoms are mainly derived from intracellular chrysolaminaran and not extracellular polysaccharides

The glucans extracted with warm water from diatoms are mainly derived from intracellular chrysolaminaran and not extracellular polysaccharides

Improved efficiency of brewer’s spent grain arabinoxylans by ultrasound-assisted extraction

Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029

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