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Analysis of Sulfate Esters by Solvolysis or Hydrolysis
Publication Name:
Current Protocols in Molecular Biology
Unit Number:
Unit 17.23
DOI:
10.1002/0471142727.mb1723s32
Online Posting Date:
May, 2001 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.
Table of Contents
- Unit Introduction
- Basic Protocol 1: Solvolysis for Release of Sulfate Esters from Glycans
- Support Protocol: 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
Materials
Basic Protocol 1: Solvolysis for Release of Sulfate Esters from Glycans
Materials
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Sulfated sample (UNIT
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Glucose-6-sulfate (Sigma)
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0.5 × 50–cm Sephadex G-25 gel-filtration column (Pharmacia Biotech)
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2-cm column of
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Pyridine
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1 M NaCl
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9:1 (v/v) dimethyl sulfoxide (DMSO; reagent grade)/methanol
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QAE-Sephadex (Pharmacia Biotech)
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Thick-walled conical glass tubes
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Heating block
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Lyophilizer or shaker-evaporator
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Additional reagents and equipment for desalting by gel-filtration chromatography (UNIT
Support Protocol: Monitoring Solvolysis Reaction
Additional Materials (also see Basic Protocol 1 )
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[
3 H]glucitol-6-sulfate: glucose-6-sulfate (Sigma) reduced with NaB[3 H]4 (UNIT -
2-cm QAE-Sephadex column (Pharmacia Biotech)
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35 mM and 200 mM NaCl
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Scintillation fluid compatible with aqueous samples
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Additional reagents and equipment for QAE-Sephadex analysis (UNIT
Alternate Protocol 1: Scale-Up of Solvolysis for Large Amounts of Material
Additional Materials (also see Basic Protocol 1 )
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[
3 H]glucitol-6-sulfate: glucose-6-sulfate (Sigma) reduced with NaB[3 H]4 (UNIT -
Dialysis tubing (3000 MWCO)
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Additional reagents and equipment for dialysis (APPENDIX
Basic Protocol 2: Acid Hydrolysis for Release of Sulfate Esters from Glycosaminoglycans
Materials
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Sample:
35 SO4 -labeled oligosaccharide or glycopeptide -
0.50 N HCl (freshly diluted from 12 N reagent grade acid)
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0.50 N NaOH (accurately titrated)
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4 M NaCl
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0.01 N HCl in 1 M NaCl (diluted from 0.50 N HCl above)
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2 mM Na
2 SO4 in 2 M KCl -
20 mM barium acetate
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Heating block with wells filled with oil
Alternate Protocol 2: Base Hydrolysis for Release of Sulfate Esters from Glycosaminoglycans
Additional Materials (also see Basic Protocol 2 )
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Sample:
35 SO4 -labeled, borohydride-reduced (UNIT -
2 N NaOH
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2 N HCl (freshly diluted from 12 N reagent-grade acid)
Looking for materials? Suppliers Guide
Figures
-
Figure 17.23.1Examples of kinetics of loss of35 SO4 from N-linked oligosaccharides of Dictyostelium discoideum. Two different types of N-linked oligosaccharides were isolated from secreted glycoproteins. (A) One type was sensitive to endo H digestion and consisted entirely of a single kinetic class, probably a 6-O sulfate ester with a t½ of 120 min. (B) The other kinetic class was released only by peptide:N-glycosidase F (PNGase F) digestion and not by endo H. It consists of two classes of sulfate esters: the major class (~80%) has a t½ similar to that of the endo H–sensitive oligosaccharide type (in panel A), but a minor class (~20%) shows a different t½ (and may be in equatorial linkage). -
Figure 17.23.2Formation of 3,6-anhydromannose by base treatment of an oligosaccharide containing mannose-6-sulfate. The formation of the anhydro sugar makes the glycosidic linkage very sensitive to acid; mild hydrolysis causes cleavage. Stronger hydrolysis degrades the usual glycosidic linkages, but leaves the anhydrosugar. When reduced with sodium borohydride, the modified sugar can be quantified to show how much mannose-6-sulfate was originally present.
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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