Analysis of Sulfate Esters by Solvolysis or Hydrolysis

Hudson H. Freeze1

1 La Jolla Cancer Research Foundation, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.23
DOI:  10.1002/0471142727.mb1723s32
Online Posting Date:  May, 2001
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Abstract

Sulfate esters are found on N‐ and O‐linked sugar chains or glycosaminoglycan (GAG) chains. Few sulfatases are available that can enzymatically remove them, so chemical procedures must be used. These procedures rely on the differential sensitivity of sulfates located in different linkages on the sugar. In comparison to the conditions used for enzymatic digestion, those used for chemical digestion are very harsh and cannot be used on protein‐bound carbohydrates (except for analytical purposes, as described here). With protein‐bound carbohydrates, for preparative purposes, the chains must first be released. This unit describes release of sulfate esters by solvolysis along with a method for monitoring the efficiency of the solvolysis reaction. An alternate procedure provides a scale‐up method for using the solvolysis reaction with large amounts of material. Also presented are techniques for both acid and basic hydrolysis to release sulfate esters.

     
 
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Table of Contents

  • Basic Protocol 1: Solvolysis for Release of Sulfate Esters from Glycans
  • Support Protocol 1: Monitoring Solvolysis Reaction
  • Alternate Protocol 1: Scale‐Up of Solvolysis for Large Amounts of Material
  • Basic Protocol 2: Acid Hydrolysis for Release of Sulfate Esters from Glycosaminoglycans
  • Alternate Protocol 2: Base Hydrolysis for Release of Sulfate Esters from Glycosaminoglycans
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Solvolysis for Release of Sulfate Esters from Glycans

  Materials
  • Sulfated sample (unit 17.1517.15), radiolabeled (unit 17.4 or unit 17.5)
  • Glucose‐6‐sulfate (Sigma)
  • 0.5 × 50–cm Sephadex G‐25 gel‐filtration column (Pharmacia Biotech)
  • 2‐cm column of recipeDowex‐50 X8 (hydrogen form; see recipe for washing procedure) packed in a Pasteur pipet plugged with glass wool
  • Pyridine
  • 1 M NaCl
  • 9:1 (v/v) dimethyl sulfoxide (DMSO; reagent grade)/methanol
  • QAE‐Sephadex (Pharmacia Biotech)
  • Thick‐walled conical glass tubes
  • Heating block
  • Lyophilizer or shaker‐evaporator
  • Additional reagents and equipment for desalting by gel‐filtration chromatography (unit 10.9)

Support Protocol 1: Monitoring Solvolysis Reaction

  • [3H]glucitol‐6‐sulfate: glucose‐6‐sulfate (Sigma) reduced with NaB[3H] 4 (unit 17.5)
  • 2‐cm QAE‐Sephadex column (Pharmacia Biotech)
  • 35 mM and 200 mM NaCl
  • Scintillation fluid compatible with aqueous samples
  • Additional reagents and equipment for QAE‐Sephadex analysis (unit 17.20)

Alternate Protocol 1: Scale‐Up of Solvolysis for Large Amounts of Material

  • [3H]glucitol‐6‐sulfate: glucose‐6‐sulfate (Sigma) reduced with NaB[3H] 4 (unit 17.5)
  • Dialysis tubing (3000 MWCO)
  • Additional reagents and equipment for dialysis ( appendix 3C)

Basic Protocol 2: Acid Hydrolysis for Release of Sulfate Esters from Glycosaminoglycans

  Materials
  • Sample: 35SO 4‐labeled oligosaccharide or glycopeptide
  • 0.50 N HCl (freshly diluted from 12 N reagent grade acid)
  • 0.50 N NaOH (accurately titrated)
  • 4 M NaCl
  • 0.01 N HCl in 1 M NaCl (diluted from 0.50 N HCl above)
  • 2 mM Na 2SO 4 in 2 M KCl
  • 20 mM barium acetate
  • Heating block with wells filled with oil

Alternate Protocol 2: Base Hydrolysis for Release of Sulfate Esters from Glycosaminoglycans

  • Sample: 35SO 4‐labeled, borohydride‐reduced (unit 17.5) oligosaccharide or glycopeptide
  • 2 N NaOH
  • 2 N HCl (freshly diluted from 12 N reagent‐grade acid)
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Figures

Videos

Literature Cited

Literature Cited
   Inoue, Y. and Nagasawa, K. 1976. Selective N‐desulfation of heparin with dimethyl sulfoxide containing water or methanol. Carbohydr. Res. 46:87‐95.
   Nagasawa, K., Inoue, Y., and Kamata, T. 1977. Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol. Carbohydr. Res. 58:47‐55.
   Freeze, H. and Wolgast, D. 1984. Structural analysis of N‐linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. J. Biol. Chem. 261:127‐133.
   Rees, D.A. 1963. A note on the characterization of carbohydrate sulphates by acid hydrolysis. Biochem J. 88:343‐346.
   Percival, E. 1978. Sulfated polysaccharides of the Rhodophycea. In Carbohydrate Sulfates. (R.G. Schweiger, ed.) pp. 213‐224. American Chemical Society, Washington, D.C.
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