Acid Hydrolysis for Release of Monosaccharides

Adriana Manzi1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.16
DOI:  10.1002/0471142727.mb1716s32
Online Posting Date:  May, 2001
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Abstract

The first step in obtaining a compositional analysis of a glycoconjugate is the release of the individual monosaccharide constituents. Mild acid hydrolysis to release fucosyl residues from glycoconjugates is described in this unit, in addition to mild acid hydrolysis to release sialic acids from glycoconjugates, along with dialysis and column chromatography procedures to purify the sialic acids. Strong acid hydrolysis to release all monosaccharides from glycoconjugates is also detailed.

     
 
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Table of Contents

  • Basic Protocol 1: Mild Acid Hydrolysis for Release of Fucose Residues
  • Basic Protocol 2: Release of Sialic Acids (Excluding 4‐O‐Acetylated Species) by Mild Acid Hydrolysis and Purification of the Product
  • Alternate Protocol 1: Release and Purification of 4‐O‐Acetylated Sialic Acids
  • Basic Protocol 3: Strong Acid Hydrolysis for Quantitative Release of Hexoses, Pentoses, Hexosamines, and Uronic Acids from Glycoconjugates
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Mild Acid Hydrolysis for Release of Fucose Residues

  Materials
  • Glycoconjugate‐containing sample to be analyzed
  • Standard: 1 µmol/ml L‐fucose (mol. wt. 164.2) in H 2O
  • 0.05 M HCl prepared from constant‐boiling HCl (sequencer‐grade, Pierce)
  • 99% methanol (anhydrous; e.g., Aldrich)
  • Small glass culture tubes or 3.5‐ml glass vials with Teflon‐lined screw caps
  • Heating block or oven
  • Nitrogen (N 2) or vacuum evaporation system: e.g., Speedvac (Savant), shaker‐evaporator (Baxter Scientific), or lyophilizer
  • Dialysis tubing (500 MWCO)
  • Additional reagents and equipment for phenol–sulfuric acid assay for monosaccharides (unit 17.9), dialysis ( appendix 3C), and compositional analysis of monosaccharides from glycoproteins (unit 17.19)

Basic Protocol 2: Release of Sialic Acids (Excluding 4‐O‐Acetylated Species) by Mild Acid Hydrolysis and Purification of the Product

  Materials
  • Glycoconjugate‐containing sample to be analyzed
  • recipeSodium formate buffer, pH 5.5 (see recipe)
  • 0.1 N H 2SO 4
  • 10 M and 2 M acetic acid
  • 1% butylated hydroxytoluene (BHT; Sigma) in ethanol
  • Dowex AG 50W‐X2 ion‐exchange resin (200 to 400 mesh, hydrogen form; Bio‐Rad)
  • Dowex AG 3‐X4A ion‐exchange resin (100 to 200 mesh, hydroxyl form; Bio‐Rad)
  • 10 mM and 1 M formic acid (ACS certified), ice‐cold
  • Dialysis tubing (1,000 and 12,000 MWCO; Spectrapor, Spectrum)
  • Heating block
  • 5‐ml glass culture tubes with Teflon‐lined screw caps
  • 0.5 × 10–cm glass chromatography columns or Pasteur pipets plugged with glass wool
  • 20‐ml glass test tubes
  • Lyophilizer or shaker‐evaporator (Baxter Scientific)
  • Additional reagents and equipment for dialysis ( appendix 3C), de‐O‐acetylation of sialic acids and TBA assay (unit 17.18), and ion‐exchange chromatography (unit 10.10)

Alternate Protocol 1: Release and Purification of 4‐O‐Acetylated Sialic Acids

  • 23.4 M (concentrated) formic acid
  • Heating block

Basic Protocol 3: Strong Acid Hydrolysis for Quantitative Release of Hexoses, Pentoses, Hexosamines, and Uronic Acids from Glycoconjugates

  Materials
  • Glycoconjugate‐containing sample to be analyzed
  • Internal standard (i.e., a monosaccharide that does not occur naturally in the sample)
  • recipeStandard mixture (see recipe)
  • 2 M and 4 M trifluoracetic acid prepared from concentrated TFA (HPLC/spectra grade sequanal quality, Pierce)
  • 99% methanol (anhydrous; e.g., Aldrich)
  • Heating block or oven
  • Small glass culture tubes or 3.5‐ml glass vials with Teflon‐lined screw caps
  • Nitrogen (N 2) or vacuum evaporation system: e.g., Speedvac (Savant) or shaker‐evaporator (Baxter Scientific)
  • Additional reagents and equipment for phenol–sulfuric acid assay for monosaccharides (unit 17.9) and compositional analysis of monosaccharides released from glycoproteins (unit 17.19)
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Literature Cited

Literature Cited
   Albersheim, P., Nevins, D.J., English, P.D., and Karr, A. 1967. A method for the analysis of sugars in plant cell‐wall polysaccharides by gas‐liquid chromatography. Carbohydr. Res. 5:340‐345.
   Biermann, C.H. 1988. Hydrolysis and other cleavages of glycosidic linkages in polysaccharides. Adv. Carbohydr. Chem. Biochem. 46:251‐271.
   Gottschalk, A. 1972. Glycoproteins: Their Composition, Structure, and Function (A. Gottschalk, ed.), 2nd ed., Part A, pp. 225‐299. Elsevier/North‐Holland, Amsterdam.
   Gyorky, G. and Houck, J.C. 1965. The determination of terminal protein‐bound fucose. Can. J. Biochem. 43:1807‐1811.
   Honda, S., Suzuki, S., Kakahi, K., Honda, A., and Takai, T. 1981. Analysis of the monosaccharide compositions of total nondialyzable urinary glycoconjugates by the dithioacetal method. J. Chromatogr. 226:341‐350.
   Kamerling, J.P. and Vliegenthart, J.F.G. 1989. Carbohydrates. In Clinical Biochemistry: Principles, Methods, Applications. Vol. 1, Mass Spectrometry (A.M. Lawson, ed.) pp. 175‐263. Walter de Gruyter & Co., Berlin.
   Keene, L., Lindberg, B., Madden, J.K., Lindberg, A.A., and Gemski, P. 1983. Structural studies of the Escherichia coli O‐antigen 25. Carbohydr. Res. 122:249‐256.
   Ludowieg, J. and Benmaman, J.D. 1967. Colorimetric differentiations of hexosamines. Anal. Biochem. 19:80‐88.
   Manzi, A.E., Dell, A., Azadi, P., and Varki, A. 1990. Studies of naturally occurring modifications of sialic acids by fast‐atom bombardment–mass spectrometry. Analysis of positional isomers by periodate cleavage. J. Biol. Chem. 265:8094‐8107.
   Neeser, J.R. and Schweizer, T.F. 1984. A quantitative determination by capillary gas‐liquid chromatography of neutral and amino sugars (as O‐methyloxime acetates), and a study on hydrolytic conditions for glycoproteins and polysaccharides in order to increase sugar recoveries. Anal. Biochem. 142:58‐67.
   Schauer, R. 1978. Biosynthesis of sialic acids. Methods Enzymol. 50:64‐89.
   Schauer, R. 1982. Chemistry, metabolism, and biological functions of sialic acids. Adv. Carbohydr. Chem. Biochem. 40:131‐234.
   Schauer, R. and Corfield, A.P. 1982. In Sialic Acids: Chemistry, Metabolism and Function (R. Schauer, ed.) pp. 51‐57. Springer‐Verlag, New York.
   Schrager, J. and Oates, M.D.G. 1968. The carbohydrate components of the hydrolysates of gastric secretion and extracts from mucuous glands of the gastric body mucosa and antrum. Biochem. J 106:523‐529.
   Varki, A. 1992. Diversity in the sialic acids. Glycobiology 2:25‐40.
   Varki, A. and Diaz, S. 1983. The release and purification of sialic acids from glycoconjugates: Methods to minimize the loss and migration of O‐acetyl groups. Anal. Biochem. 137:236‐247.
   Varki, A. and Kornfeld, S. 1980. An autosomal dominant gene regulates the extent of 9‐O ‐actylation of murine erythrocyte sialic acids. J. Exp. Med. 152:532‐544.
Key References
   Biermann, 1988. See above.
  These references discuss the mechanisms of acid hydrolysis of the different monosaccharides, and the influence of structure in determining alternative mechanisms. They also review many different hydrolysis conditions, and illustrate application of particular conditions to different types of glycoconjugates and the results obtained.
   Gottschalk, 1972. See above.
  Describes the parameters that must be varied to arrive at the optimal conditions used in the method described here.
   Varki and Diaz, 1983. See above.
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