Direct Chemical Analysis of Glycoconjugates for Carbohydrates

Adriana Manzi1, Jeffrey Esko2

1 University of California San Diego, La Jolla, California, 2 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.9
DOI:  10.1002/0471142727.mb1709s32
Online Posting Date:  May, 2001
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Abstract

This unit presents protocols for detection of different sugars bound to glycoconjugates. The solution containing the carbohydrate material is treated with a specific reagent, generating a colored reaction product that can be detected spectrophotometrically. For each assay, the absorbance of the colored solution is proportional to the amount of sugar present in the glycoconjugate.

     
 
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Table of Contents

  • Basic Protocol 1: Phenol–Sulfuric Acid Assay for Hexoses and Pentoses
  • Reagents and Solutions
  • Commentary
  • Basic Protocol 2: Ferric Orcinol Assay for Sialic Acids
  • Reagents and Solutions
  • Commentary
  • Basic Protocol 3: MBTH Assay for Hexosamines and Acetylhexosamines
  • Commentary
  • Basic Protocol 4: Carbazole Assay for Uronic Acids
  • Alternate Protocol 1: Meta‐Hydroxybiphenyl Assay for Uronic Acids
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Phenol–Sulfuric Acid Assay for Hexoses and Pentoses

  Materials
  • Sample to be assayed
  • Monosaccharide standard: e.g., D‐(+)‐galactose (Sigma, purified‐grade anhydrous crystals; )
  • recipe5% (w/v) phenol (see recipe)
  • Concentrated sulfuric acid (ACS reagent‐grade)
  • Thick‐walled, 16 × 125–mm Pyrex test tubes, either brand new and washed with distilled water, or acid washed
  • Automatic glass dispenser or glass pipet with a portion of the tip removed for rapid flow, suitable for use with concentrated sulfuric acid
  • 1.0‐ml glass cuvettes (1‐cm path length)
  • Visible‐light spectrophotometer

Basic Protocol 2: Ferric Orcinol Assay for Sialic Acids

  Materials
  • Sample to be assayed
  • Sialic acid standard: 1 mM N‐acetylneuraminic acid (Neu5Ac; Boehringer Mannheim; store frozen at −20°C)
  • recipeBial reagent (see recipe)
  • Isoamyl alcohol
  • Thick‐walled 16 × 125–mm Pyrex test tubes
  • Heating block or boiling water bath
  • Glass marbles
  • 1.0‐ml glass cuvettes (1‐cm path length)
  • Visible‐light spectrophotometer

Basic Protocol 3: MBTH Assay for Hexosamines and Acetylhexosamines

  Materials
  • Sample to be analyzed
  • 1 M HCl (ACS reagent‐grade)
  • Hexosamine standard: 1 mM N‐acetyl‐D‐glucosamine or N‐acetyl‐D‐galactosamine (Sigma)
  • 2.5% (w/v) sodium nitrite (Sigma; store in dark bottle at room temperature; prepare fresh weekly)
  • 12.5% (w/v) ammonium sulfamate (ACS reagent‐grade; store ≥2 months at room temperature)
  • 0.25% (w/v) 3‐methyl‐2‐benzothiazolinone hydrazone hydrochloride (MBTH; Kodak; store in dark bottle at 4°C and replace if yellowish)
  • 0.5% (w/v) ferric chloride (certified ACS reagent‐grade; store indefinitely at room temperature)
  • 13 × 100–mm Pyrex test tubes with Teflon‐lined screw caps
  • Heating block, 110°C
  • Water baths, room temperature and 37°C
  • 1.0‐ml glass cuvettes (1‐cm path length)
  • Visible‐wavelength spectrophotometer

Basic Protocol 4: Carbazole Assay for Uronic Acids

  Materials
  • Sample to be assayed
  • Uronic acid standard: recipe1 mM glucuronolactone (see recipe)
  • 4 M ammonium sulfamate (Sigma)
  • recipe25 mM sodium tetraborate in sulfuric acid (see recipe)
  • 0.1% (w/v) carbazole (Kodak) in 95% ethanol (store at 4°C)
  • 16 × 125–mm Pyrex tubes (open, glass‐stoppered, or screw‐cap culture tubes fitted with Teflon‐lined caps)
  • Water bath, 100°C, or heating block
  • Disposable glass or plastic 1.0‐ml cuvettes
  • UV/VIS spectrophotometer

Alternate Protocol 1: Meta‐Hydroxybiphenyl Assay for Uronic Acids

  • 0.15% (w/v) meta‐hydroxybiphenyl (Kodak) in 0.5% (w/v) NaOH
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Figures

Videos

Literature Cited

   Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350‐356.
  Original description of this technique, containing the most complete study to date of the reactivity of different types of carbohydrates.
   Klenk, E. and Langerbeins, H. 1941. Uber die Verteilung der Neuraminsaure im Gehirn (Mit einer Mikromethode zur quantitativen Bestimmung der Substanz im Nervengewebe). Hoppe‐Seyler's Z. Physiol. Chem. 270:185‐193.
   Schauer, R. 1978. Characterization of sialic acids. Methods Enzymol. 50:64‐89.
   Spiro, R.G. 1966. Analysis of sugars found in glycoproteins. Methods Enzymol. 8:3‐26.
   Svennerholm, L. 1963. Sialic acids and derivatives: Estimation by the ion‐exchange method. Methods Enzymol. 6:459‐462.
   Veh, R.W., Corfield, A.P., Sander, M., and Schauer, R. 1977. Neuraminic acid–specific modification and tritium labelling of gangliosides. Biochim. Biophys. Acta 486:145‐160.
   Smith, R.L. and Gilkerson, E. 1979. Quantitation of glycosaminoglycan hexosamine using 3‐methyl‐2‐benzothiazolone hydrazone hydrochloride. Anal. Biochem. 98:478‐480.
   Tsuji, A., Kinoshita, T., and Hoshino, M. 1969a. Microdetermination of hexosamines. Chem. Pharm. Bull. 17:217‐218.
   Tsuji, A., Kinoshita, T., and Hoshino, M. 1969b. Analytical chemical studies on amino sugars. II. Determination of hexosamines using 3‐methyl‐2‐benzothiazolone hydrazone hydrochloride. Chem. Pharm. Bull. 17:1505‐1510.
   Bitter, T. and Muir, H.M. 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4:330‐334.
   Blumenkrantz, N. and Asboe‐Hansen, G. 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54:484‐489.
   Dische, Z. 1947. A new specific color reaction of hexuronic acids. J. Biol. Chem. 167:189‐198.
   Filisetti‐Cozzi, T.M. and Carpita, N.C. 1991. Measurement of uronic acids without interference from neutral sugars. Anal. Biochem. 197:157‐162.
   Galambos, J.T. 1967. The reaction of carbazole with carbohydrates. I. Effect of borate and sulfamate on the carbazole color of sugars. Anal. Biochem. 19:119‐132.
   Kosakai, M. and Yosizawa, Z. 1978. Study on the factors yielding high color in the carbazole reaction with hexuronic acid–containing substances. J. Biochem. 84:779‐785.
Key References
   Schauer, R. 1978. See above.
  Describes the method and comments on its specificity and sensitivity.
   Smith, R.L. and Gilkerson, E. 1979. See above.
  Describes the simplified protocol and its application to glycosaminoglycans.
   Bitter and Muir, 1962. See above.
  These two references describe the most recent modifications of the methods.
   Filisetti‐Cozzi and Carpita 1991. See above.
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