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A practical guide to structural analysis of carbohydrates

Introduction

This guide is a collection of standarized procedures used at Stockholm University in the laboratory of Prof. Per-Erik Jansson (now at Klinisk Forskningscentrum/Karolinska Institutet. The original manuscript, excluding figures, was translated to HTML by Roland Stenutz.
There are still many omissions and errors so be careful when using it and do not rely its accuracy.
Some of the reagents reffered to are highly toxic and/or corrosive and must be handled with extreme care. In most cases normal laboratory practice will suffice (fume hood, lab coat, safety goggles).
Corrections and additions are most welcome and should be addressed to Roland Stenutz, roland.stenutz@organ.su.se.
If you find this guide useful please cite it as:
R. Stenutz, P.-E. Jansson and G. Widmalm; "A practical guide to structural analysis of carbohydrates"
http://www.casper.organ.su.se/sop and date of access.

Index

  1. Component analysis
  2. Specific degradations
  3. Modification of polysaccharides
  4. Purification of polysaccharides
  5. Preparation of reagents
  6. Chromatography
  7. Web resources

Component analysis

  1. Sugar analysis
    1. - using TFA
    2. - using TFA and morpholinoborane
    3. - using anhydrous hydrogen fluoride
    4. - using methyl glycosides
  2. Methylation analysis using partially methylated alditol acetates (PMAAs)
    1. - with dimsyl sodium/methyl iodide (CH3SOCH2-Na+) (of LOS)
    2. - with sodium hydroxide/methyl iodide
    3. - with methyl triflate
    4. - including carboxyl reduction of the native polysaccharide
    5. - including carboxyl reduction of the methylated polysaccharide
  3. Determination of absolute configuration using butyl glycosides
    1. - using silylated butyl glycosides
    2. - using acetylated butyl glycosides
  4. Fatty acid analysis using the methyl esters (total content)
  5. Protein estimation
  6. Colour reactions using a chromophoric reagent
  7. Phosphorous determination
  8. Kdo determination
  9. RNA determination
  10. GlcNAc determination
  11. Alditol trifluoroacetates

Specific degradations

  1. Smith degradation
  2. Uronic acid degradation
  3. Acetolysis
  4. N-deacylation by trifluoroacetolysis, followed by deamination or hydrolysis
  5. N-deacylation by alkali in DMSO, followed by deamination or hydrolysis

Modification of polysaccharides

  1. Delipidation of LPS
  2. Peracetylation
  3. N-acetylation
  4. N-deacylation
  5. O-deacylation
  6. Dephosphorylation
  7. Hydrazinolysis

Purification of polysaccharides

  1. Phenol extraction
  2. Phenol-Chloroform-Petroleum ether extraction (PCP-extraction)
  3. Precipitation of acidic polysaccharides with cetyltrimethyl ammonium chloride (Cetavlon)
  4. Precipitation with ethanol
  5. RNAse treatment
  6. Proteinase treatment
  7. Removal of phospholipids from LPS

Preparation of reagents

  1. Preparation of dimsyl sodium

Chromatography

  1. Biogel P4
  2. Sephadex G50
  3. Superdex
  4. DEAE-Sepharose
  5. CM-Sepharose

Web resources

  1. Mass spectra of Partially Methylated Alditol Acetates
  2. Carbohydrate structure database (CarbBank)
  3. Carbohydrate NMR database (Sugabase)
  4. Calculation of 3JH,H coupling constants
  5. Calculation of coupling constants other than 3JH,H
  6. CASPER - Simulation and interpretation of polysaccharide NMR
  7. SWEET - 3D-structure of carbohydrates
  8. SweetDB - A database linking structures and bibliographic information
  9. WWW resources - A list

Appendix - Carbohydrate structures

  1. The aldoses
  2. The ketoses
  3. NMR data for some pyranoses
Uppdaterat: Dec 8, 2004 | Redaktör | Tekniska frågor | Om www.su.se