Chemical Labeling of Carbohydrates by Oxidation and Sodium Borohydride Reduction

Minoru Fukuda1

1 La Jolla Cancer Research Foundation, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.5
DOI:  10.1002/0471142727.mb1705s26
Online Posting Date:  May, 2001
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Abstract

This unit describes a collection of methods for chemical labeling of carbohydrates (free oligosaccharides or oligosaccharides conjugated to proteins, peptides, or lipids) by oxidation followed by reduction or by direct reduction. Oligosaccharides can be labeled with either radioisotopes or nonradioactive fluorescent molecules. These labelings allow one to follow the oligosaccharides during chromatography and in cells if labeled by fluorescent molecules. Selective oxidation with mild periodate followed by reduction with tritiated sodium borohydride results in selective radiolabeling of sialic acid residues on oligosaccharides or glycoproteins. Alternatively, treatment of samples with galactose oxidase results in oxidation of galactose or N‐acetylgalactosamine residues at nonreducing termini, rendering these residues suceptible to labeling with NaB[3H]4. Oxidized glycoconjugates can also be labeled using the fluorescent probe lucifer yellow CH. Free oligosaccharides can be labeled by reduction with NaB[3H]4. An additional protocol describes the release and simultaneous labeling of O‐glycan oligosaccharides by alkaline beta‐elimination in the presence of NaB[3H]4.

     
 
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Table of Contents

  • Basic Protocol 1: Radiolabeling Oligosaccharides After Mild Periodate Oxidation
  • Alternate Protocol 1: Radiolabeling Oligosaccharides After Galactose Oxidase Treatment
  • Alternate Protocol 2: Fluorescence Labeling Oligosaccharides After Periodate or Galactose Oxidase Treatment
  • Basic Protocol 2: Radiolabeling Free Oligosaccharides
  • Alternate Protocol 3: Radiolabeling O‐Glycan Oligosaccharides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Radiolabeling Oligosaccharides After Mild Periodate Oxidation

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • Oligosaccharide or glycopeptide containing sialic acids, desalted
  • 0.2 mM sodium periodate (prepare fresh at room temperature and keep on ice)
  • 10 mM sodium acetate, pH 5.5
  • 10 mM glycerol (store at 4°C)
  • 0.2 M sodium borate buffer, pH 9.5 (unit 11.16; adjust quantities of boric acid and 10 M NaOH appropriately)
  • recipe0.5 mCi/µl tritiated sodium borohydride (NaB[3H] 4; 25 to 30 Ci/mmol) in 0.01 M NaOH (see recipe)
  • 0.2 M sodium borohydride (NaBH 4)/0.2 M sodium borate buffer, pH 9.5
  • 1 M acetic acid in methanol
  • Nitrogen (N 2) stream
  • Methanol
  • 7‐ml conical glass (Pyrex) test tubes with Teflon‐covered caps
  • 1.0 × 40–cm Sephadex G‐15 or G‐25 column in water (unit 10.9), calibrated to identify void volume
  • 25° to 30°C water bath
CAUTION: Steps to must be performed in a well‐vented fume hood to prevent radioactive contamination. Excess NaB[3H] 4 should be stored away in a radioactive waste bottle for appropriate disposal.

Alternate Protocol 1: Radiolabeling Oligosaccharides After Galactose Oxidase Treatment

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • 1 U/µl galactose oxidase (partially purified, Sigma; store aliquots in PBS at −20°C)
  • 10 mM sodium phosphate buffer, pH 7.0
  • 15‐ml conical glass centrifuge tube
CAUTION: Steps to must be performed in a well‐vented fume hood to prevent radioactive contamination.

Alternate Protocol 2: Fluorescence Labeling Oligosaccharides After Periodate or Galactose Oxidase Treatment

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Lucifer yellow CH (Aldrich)
  • 0.1 M sodium cyanoborohydride (NaBH 3CN)
  • 0.5 × 15–cm Sephadex G‐15 and 1.0 × 25–cm Sephadex G‐25 columns in water (unit 10.9), calibrated
  • 25°C water bath

Basic Protocol 2: Radiolabeling Free Oligosaccharides

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • Free oligosaccharides (units 17.15 & 17.12)
  • 0.3 M sodium borate buffer, pH 9.5 (unit 11.16; adjust quantities of boric acid and 10 M NaOH appropriately)
  • recipe0.5 mCi/µl tritiated sodium borohydride (NaB[3H] 4; 25 to 35 Ci/mmol) in 0.05 M NaOH (see recipe)
  • 1 M sodium borohydride (NaBH 4)
  • 1 M acetic acid in methanol
  • Nitrogen (N 2) stream
  • Methanol
  • 7‐ml conical glass (Pyrex) tubes
  • 0.5 × 25–cm Sephadex G‐25 or G‐15 column (unit 10.9), calibrated to determine void volume
  • Additional reagents and equipment for gel‐filtration chromatography (unit 10.9)
CAUTION: Steps to must be performed in a well‐vented fume hood to prevent radioactive contamination.

Alternate Protocol 3: Radiolabeling O‐Glycan Oligosaccharides

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • 100 to 1000 µg glycopeptides or ≤5 mg glycoproteins
  • recipe0.1 to 0.2 mCi/µl tritiated sodium borohydride (NaB[3H] 4; 25 to 30 Ci/mmol) in 1 M NaBH 4/0.05 M NaOH (see recipe)
  • 1 M acetic acid in methanol
  • Methanol
  • 30° to 35°C and 45°C water baths
CAUTION: Steps to must be performed in a well‐vented fume hood to avoid radioactive contamination.
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Figures

Videos

Literature Cited

Literature Cited
   Amano, J. and Kobata, A. 1989. Quantitative conversion of mucin‐type sugar chains to radioactive oligosaccharides. Methods Enzymol. 179:261‐270.
   Fukuda, M., Carlsson, S.R., Klock, J.C., and Dell, A. 1986. Structures of O‐linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells. J. Biol. Chem. 261:12796‐12806.
   Morell, A.G. Ashwell, G. 1972. Tritium labeling of glycoproteins that contain terminal galactose residue. Methods Enzymol. 28:205‐208.
   Sasaki, H., Bothner, B., Dell, A., and Fukada, M. 1987. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J. Biol. Chem. 262:12059‐12076.
   Spiegel, S. 1987. Fluorescent gangliosides. Methods Enzymol. 138:313‐318.
   Takasaki, S. and Kobata, A. 1978. Microdetermination of sugar composition by radioisotope labeling. Methods Enzymol. 50:50‐54.
   Wilchek, M., Spiegel, S., and Spiegel, Y. 1980. Fluorescent reagents for the labeling of glycoconjugates in solution and on cell surfaces. Biochem. Biophys. Res. Commun. 92:1215‐1222.
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