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Chemical Labeling of Carbohydrates by Oxidation and Sodium Borohydride Reduction
Publication Name:
Current Protocols in Molecular Biology
Unit Number:
Unit 17.5
DOI:
10.1002/0471142727.mb1705s26
Online Posting Date:
May, 2001 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[
Table of Contents
- Unit Introduction
- Basic Protocol: Radiolabeling Oligosaccharides After Mild Periodate Oxidation
- Alternate Protocol: Radiolabeling Oligosaccharides After Galactose Oxidase Treatment
- Alternate Protocol: Fluorescence Labeling Oligosaccharides After Periodate or Galactose Oxidase Treatment
- Basic Protocol: Radiolabeling Free Oligosaccharides
- Alternate Protocol: Radiolabeling O-Glycan Oligosaccharides
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
Materials
Basic Protocol: Radiolabeling Oligosaccharides After Mild Periodate Oxidation
Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see APPENDIX 2 ; for suppliers, see APPENDIX 4 .
- 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
3 H]4 ; 25 to 30 Ci/mmol) in 0.01 M NaOH- 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
- 25° to 30°C water bath
CAUTION: Steps to must be performed in a well-vented fume hood to prevent radioactive contamination. Excess NaB[3 H]4 should be stored away in a radioactive waste bottle for appropriate disposal.
Alternate Protocol: Radiolabeling Oligosaccharides After Galactose Oxidase Treatment
Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see APPENDIX 2 ; for suppliers, see APPENDIX 4 .
- 1 U/µl galactose oxidase (partially purified, Sigma; store aliquots in PBS at –20°C)
- 15-ml conical glass centrifuge tube
CAUTION: Steps to must be performed in a well-vented fume hood to prevent radioactive contamination.
Alternate Protocol: Fluorescence Labeling Oligosaccharides After Periodate or Galactose Oxidase Treatment
Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see APPENDIX 2 ; for suppliers, see APPENDIX 4 .
- Lucifer yellow CH (Aldrich)
- 0.1 M sodium cyanoborohydride (NaBH
3 CN) - 0.5 × 15–cm Sephadex G-15 and 1.0 × 25–cm Sephadex G-25 columns in water (UNIT
- 25°C water bath
Basic Protocol: Radiolabeling Free Oligosaccharides
Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see APPENDIX 2 ; for suppliers, see APPENDIX 4 .
- Free oligosaccharides (UNITS
- 0.3 M sodium borate buffer, pH 9.5 (UNIT
3 H]4 ; 25 to 35 Ci/mmol) in 0.05 M NaOH- 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
- Additional reagents and equipment for gel-filtration chromatography (UNIT
CAUTION: Steps to must be performed in a well-vented fume hood to prevent radioactive contamination.
Alternate Protocol: Radiolabeling O-Glycan Oligosaccharides
Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see APPENDIX 2 ; for suppliers, see APPENDIX 4 .
- 100 to 1000 µg glycopeptides or £5 mg glycoproteins
3 H]4 ; 25 to 30 Ci/mmol) in 1 M NaBH4 /0.05 M NaOH- 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.
Looking for materials? Suppliers Guide
Figures
-
Figure 17.5.1Labeling oligosaccharides by periodate oxidation followed by NaB[3 H]4 reduction. -
Figure 17.5.2Labeling oligosaccharides by galactose oxidase treatment followed by NaB[3 H]4 reduction.
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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The recipe for the stock solution of tritiated sodium borohydride is as follows:
Tritiated sodium borohydride (NaB[3H]4), 0.5 mCi/µl
Dissolve 100 mCi NaB[3H]4 (highest available specific activity, usually 25 to 35 Ci/mmol) in 200 µl ice-cold 0.01 or 0.05 M NaOH.
Store in 10-µl aliquots for several months at -70°C until use.
To make NaB[3H]4 in 1 M NaBH4/0.05M NaOH, dissolve solid NaBH4 in tritiated solution in 0.05 M NaOH.
Alternatively, NaB[3H]4 can be dissolved in freshly distilled anhydrous dimethylformamide and stored at -20°C in well-sealed tubes. Some investigators report stability for longer periods than with NaOH solutions.
I wish to know about the stock solution of sodium borohydride.
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