Nitrous Acid Degradation of Glycosaminoglycans

H. Edward Conrad1

1 University of Illinois, Urbana, Illinois
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.22A
DOI:  10.1002/0471142727.mb1722as32
Online Posting Date:  May, 2001
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Abstract

Glycosaminoglycans (GAGs) are made up of disaccharide units that are distinguished from each other by the monosaccharide units of which they are composed and by the degree and position of sulfation. These disaccharide units represent the monomeric units of the GAG; thus, measurement of the disaccharide composition of a GAG represents the first step in the characterization of the polymer. In this unit, cleavage of the glycosidic bonds of the N‐sulfated GlcN residues in heparin and heparan sulfate is described, in addition to cleavage of the bonds between the N‐acetylated amino sugar residues in heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate, and hyaluronic acid. Using these procedures involving, all GAGs can be converted completely to their constituent disaccharides and reduced with NaB[3H]4) to yield labeled disaccharides that can be assayed qualitatively or quantitatively. The procedure may also be used to analyze metabolically labeled GAGs (with or without the use of NaB[3H]4).

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • recipeReference standard (see recipe)
  • Unknown sample suspected of containing N‐sulfated or N‐acetylated GAG
  • recipeInternal standard (see recipe)
  • recipeNitrous acid reagent, pH 1.5 (see recipe)
  • 1 M Na 2CO 3
  • 10 µg/µl hydrazine sulfate in anhydrous hydrazine
  • 3 M H 2SO 4
  • recipeNitrous acid reagent, pH 4.0 (see recipe)
  • 0.5 M tritiated sodium borohydride (NaB[3H] 4; ∼500 mCi/mmol) in 0.1 M NaOH
  • 100‐µl Reacti‐Vials (Pierce)
  • Sand bath: heating block (e.g., Pierce) with wells filled with sand
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Figures

Videos

Literature Cited

   Conrad, H.E., Varboncoeur, E., and James, M.E. 1973. Qualitative and quantitative analysis of reducing carbohydrates by radiochromatography on ion exchange papers. Anal. Biochem. 51:486‐500.
   Shively, J.E. and Conrad, H.E. 1976. Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15:3932‐3942.
Key Reference
   Guo, Y. and Conrad, H.E. 1989. The disaccharide composition of heparins and heparan sulfates. Anal. Biochem. 176:96‐104.
  These references describe the basic procedures in this unit and the background for those procedures.
   Shaklee, P.N. and Conrad, H.E. 1984. Hydrazinolysis of heparin and other glycosaminoglycans. Biochemistry 217:187‐197.
   Shaklee, P.N. and Conrad, H.E. 1986. The disaccharides formed by deaminative cleavage of N‐deacetylated glycosaminoglycans. Biochem. J. 235:225‐236.
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