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Composition of Labeled Monosaccharides from Glycosaminoglycans

H. Edward Conrad1

1University of Illinois, Urbana, Illinois

Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 17.19B
DOI: 
10.1002/0471142727.mb1719bs32
Online Posting Date: 
May, 2001
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Abstract

Proteoglycans can be metabolically labeled with [3H]glucosamine to incorporate label into glucosamine and galactosamine. Glycosaminoglycans can be released from the proteoglycans and the distribution of labeled glucosamine and galactosamine in the isolated glycosaminoglycans can be determined. A series of acid treatments yields a mixture of disaccharides (uronosyl-anhydro sugars), free uronic acids, and free anhydro sugars, which are then reduced and separated by paper chromatography. The amount of each labeled component is quantified by scintillation counting. This procedure can also be used to determine the composition of glycosaminoglycans that have not been labeled metabolically. Samples of unlabeled glycosaminoglycans can be treated in the same manner and the hydrolysis/deamination mixture can be reduced with NaB3H4 to give stoichiometric 3H labeling of each reducing sugar component of the mixture.

     
 
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Table of Contents

  • Basic Protocol
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Tables
     
 
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Materials

 Basic Protocol
 Materials
  • Glycosaminoglycan sample (optionally, metabolically labeled with [3 H]glucosamine; UNIT 17.4)
  • 20 µCi/ml [l4C]glucose solution (>40 mCi/mmol; optional internal standard)
  • 3 M and 0.5 M H2SO4
  • White mineral oil
  • 5.5 M NaNO2
  • 1 M Na2CO3
  • 0.25 M NaBH4 or 0.25 M ~500 mCi/mmol NaB3H4, in 0.25 M NaOH
  • 0.1 M and 1 M NaOH
  • Paper chromatography System 1 and System 2 (see recipes)
  • Scintillation cocktail (see recipe)
  • 6 × 150–mm test tubes
  • Sand baths: heating elements (Pierce) filled with sand, 99° and 50°C
  • Hamilton syringe
  • Whatman no. 3 chromatography paper (grade 3 Chr)
  • Whatman cellulose phosphate chromatography paper (grade P 81)
  • Paper chromatography jars for descending chromatography
  • Scintillation counter suitable for dual-label counting
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Literature Cited

 Literature Cited
    Bienkowski, M.J. and Conrad, H.E. 1985. Structural characterization of the oligosaccharides formed by depolymerization of heparin with nitrous acid. J. Biol. Chem. 260:356-365.
    Conrad, H.E. 1980. The acid lability ofthe glycosidic bonds of L-iduronic acid residues in glycosaminoglycans. Biochem. J. 191:355-363.
    Shively, J.E. and Conrad, H.E. 1970. Stoichiometry ofthe nitrous acid deaminative cleavage of model amino sugar glycosides and glycosaminoglycuronans. Biochemistry 9:33-41.
    Shively, J.E. and Conrad, H.E. 1976. Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15:3932-3942.
 Key Reference
    Conrad, 1980. See above.

Describes the basic procedure used here.

     
 
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