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Analysis of Monosaccharides

Adriana E. Manzi1,  Leland D. Powell1,  Ajit Varki1

1University of California San Diego, La Jolla, California

Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
UNIT 17.18
DOI: 
10.1002/0471142727.mb1718s32
Online Posting Date: 
May, 2001
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Abstract

This unit presents methods for assaying sialic acids, reducing sugars, and hexosamines. The BCA assay detects free reducing terminii in sugars released from glycoconjugates by appropriate treatments. Assays employing Ehrlich reagent (DMAB) detect hexosamines and N-acetylhexosamines, including a method for hydrolyzing the glycosidic linkages of the hexosamines and a method for re-N-acetylation. The TBA and DMB assays can be used to quantitate and fractionate free forms of many types of sialic acids. Techniques for liberating the sialic acids from the parent glycoconjugates are also provided.

     
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Table of Contents

  • SECTION V: Analysis of Saccharides Released from Glycoconjugates
  • Unit Introduction
  • Basic Protocol 1: BCA Assay for Reducing Sugars with Spectrophotometric Detection
  • Reagents and Solutions
  • Commentary
  • Basic Protocol 2: Assays for Free Hexosamines with Spectrophotometric Detection
  • Alternate Protocol: Acidic DMAB (Morgan-Elson) Assay for Free N-Acetylhexosamines
  • Support Protocol 1: RE-N-Acetylation of Free Hexosamines
  • Reagents and Solutions
  • Commentary
  • Basic Protocol 3: Thiobarbituric Acid Assay for Sialic Acids with Spectrophotometric or HPLC Detection
  • Support Protocol 2: DE-O-Acetylation of Sialic Acids
  • Support Protocol 3: Acid Release of DE-O-Acetylated Sialic Acids
  • Reagents and Solutions
  • Commentary
  • Basic Protocol 4: DMB Assay for Sialic Acids with HPLC Detection
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
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Materials

Basic Protocol 1: BCA Assay for Reducing Sugars with Spectrophotometric Detection

 Materials
  • Sample in water or aqueous buffer
  • Standard: e.g., glucosamine hydrochloride or N-acetyl-D-glucosamine (both available from Sigma)
  • Bovine serum albumin (BSA)
  • BCA reagent (reagent C; see recipe)
  • 16 × 115–mm Pyrex test tubes
  • Clean glass marbles
  • Heating block, 80°C
  • Spectrophotometer and 1.0-ml glass cuvettes

Basic Protocol 2: Assays for Free Hexosamines with Spectrophotometric Detection

 Materials
  • Hexosamine-containing sample (³6 µg hexosamine) in aqueous solution
  • 1 mM hexosamine standard stock solution(s): N-acetylglucosamine and/or N-acetylgalactosamine (see recipe)
  • 4 M HCl (diluted from ACS reagent-grade concentrated HCl)
  • Acetylacetone reagent (see recipe)
  • 95% ethanol
  • Ehrlich reagent (DMAB)/HCl (see recipe)
  • 13 × 100–mm Pyrex test tubes with Teflon-lined screw caps
  • Nitrogen or vacuum evaporation system (Speedvac or shaker-evaporator)
  • Heating block or oven
  • Boiling water bath
  • Spectrophotometer and 1.0-ml glass cuvettes

Alternate Protocol: Acidic DMAB (Morgan-Elson) Assay for Free N-Acetylhexosamines

 Additional Materials (also see Basic Protocol 2)
  • 20 mM potassium tetraborate
  • Ehrlich reagent (DMAB)/HCl/acetic acid (see recipe)

Support Protocol 1: RE-N-Acetylation of Free Hexosamines

 Additional Materials (also see Basic Protocol 2)
  • 1.5% (v/v) acetic anhydride (99%, Aldrich) in acetone (HPLC grade, Fisher)
  • Aqueous sample containing hexosamines liberated from glycosidic linkages by acid hydrolysis, and glucosamine and galactosamine standards treated in the same fashion (see Basic Protocol 2, step )

Basic Protocol 3: Thiobarbituric Acid Assay for Sialic Acids with Spectrophotometric or HPLC Detection

 Materials
  • Sialic acid–containing sample in solution
  • Periodate reagent (see recipe)
  • 1 mM N-acetylneuraminic acid (Neu5Ac; store at –20°C; stable indefinitely)
  • 1 mM 2-deoxyribose (optional; store at –20°C; stable indefinitely)
  • Arsenite reagent (see recipe)
  • TBA reagent (see recipe)
  • Cyclohexanone (for spectrophotometric detection)
  • HPLC elution buffer (see recipe; for HPLC detetion)
  • Heating block, 100°C
  • For spectrophotometric detection:
  •     New 12 × 75–mm glass tubes
  •     Clean glass marbles
  •     Tabletop centrifuge
  •     Spectrophotometer and 1.0-ml glass cuvettes
  • For HPLC detection:
  •     0.4 × 25–cm C18 reversed-phase HPLC column
  •     Equipment for HPLC (UNIT 10.12)
  •     HPLC UV detector with flow detector cell of volume <20 µl, and integrating chart recorder if available

Support Protocol 2: DE-O-Acetylation of Sialic Acids

 Materials
  • Sialic acid–containing sample in solution
  • 0.2 M NaOH (store at room temperature)
  • 0.9 M H2SO4 (for acid release) or 0.2 M acetic acid (for enzymatic release)

Support Protocol 3: Acid Release of DE-O-Acetylated Sialic Acids

 Materials
  • Sialic acid–containing sample in solution, preferably de-O-acetylated (see Support Protocol 2)
  • 0.2 M H2SO4
  • 1% (w/v) BHT in ethanol (see recipe)
  • Clean glass marbles
  • Heating block, 80°C

Basic Protocol 4: DMB Assay for Sialic Acids with HPLC Detection

 Materials
  • N-acetylneuraminic acid (Neu5Ac, MW 309.3; Boehringer Mannheim)
  • Standard mixture: sialic acids prepared from bovine submaxillary mucin (BSM) using the procedure in UNIT 17.16, or mixture of 100 pmol each Neu5Ac and N-glycolylneuraminic acid (Neu5Gc, MW 325.3; Sigma)
  • Sample containing mixture of free sialic acids in solution (5 to 1000 pmol; UNIT 17.16 or UNIT 17.12)
  • DMB reagent (see recipe)
  • Acetonitrile (HPLC grade)
  • 50% methanol (HPLC grade) in Milli-Q-purified water
  • Milli-Q-purified water: deionized water passed through a five-stage Milli-Q Plus system (Millipore)
  • Helium (for HPLC apparatus)
  • Heating block, 50°C
  • HPLC apparatus (preferably ternary system) including pump and fluorescent detector (UNIT 10.12)
  • TSK gel ODS-120T column (250 mm × 4.6 mm i.d., 5 µm particle size; TosoHass)
  • Guard cartridge TSK gel ODS-120T (1.5 × 3.2 mm)
  • Filtering unit with 0.45-µm Nylon 66 membranes (Alltech)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 17.18.1
    Reaction of sialic acids with 1,2-diamino-4,5-methylenedioxybenzene (DMB) to form fluorescent derivatives.

    View Image
  • Figure 17.18.2
    HPLC profile (fluorescence vs. time) of the DMB derivatives of total sialic acids released from BSM (bovine submaxillary mucin) with 2 M acetic acid and purified according to the protocol described in UNIT 17.16.

    View Image

Literature Cited

 Literature Cited
    Doner, L.W. and Irwin, P.L. 1992. Assay of reducing end-groups in oligosaccharide homologues with 2,2¢-bicinchoninate. Anal. Biochem. 202:50-53.
    Folin, O. and Malmros, H. 1929. An improved form of Folin's micro method for blood sugar determinations. J. Biol. Chem. 83:115.
    McFeeters, R.F. 1980. A manual method for reducing sugar determinations with 2,2¢-bicinchoninate reagent. Anal. Biochem. 103:302-306.
    Mopper, K. and Gindler, E.M. 1973. A new noncorrosive dye reagent for automatic sugar chromatography. Anal. Biochem. 56:440-442.
    Park, J.T. and Johnson, M.J. 1949. A submicrodetermination of glucose. J. Biol. Chem. 181:149-151.
    Sinner, M. and Puls, J. 1978. Noncorrosive dye reagent for detection of reducing sugars in borate complex ion-exchange chromatography. J. Chromatogr. 156:197-204.
    Waffenschmidt, S. and Jaenicke, L. 1987. Assay of reducing sugars in the nanomole range with 2,2¢ -bicinchoninate. Anal. Biochem. 165:337-340.
    Chaplin, M.F. 1986. Monosaccharides. In Carbohydrate Analysis: A Practical Approach (M.F. Chaplin and J.F. Kennedy, eds.) pp. 1-36. IRL Press, Oxford.
    Elson, L.A. and Morgan, W.T.J. 1933. A colorimetric method for the determination of glucosamine and chondrosamine. Biochem. J. 27:1824-1828.
    Foster, A.B. and Horton, D.H. 1959. Aspects of the chemistry of the amino sugars. Adv. Carbohydr. Chem. 14:213-281.
    Levvy, G.A. and McAllan, A. 1959. The N-acetylation and estimation of hexosamines. Biochem. J. 73:127-132.
    Ludowieg, J. and Benmaman, J.D. 1967. Colorimetric differentiation of hexosamines. Anal. Biochem. 19:80-88.
    Montreuil, J., Bouquelet, S., Debray, H., Fournet, B., Spik, G., and Strecker, G. 1986. Glycoproteins. In Carbohydrate Analysis: A Practical Approach. (M.F. Chaplin and J.F. Kennedy, eds.) pp. 143-204. IRL Press, Oxford.
    Morgan, W.T.J. and Elson, L.A. 1934. A colorimetric method for the determination of N-acetylglucosamine and N-acetylchondrosamine. Biochem. J. 27:988-995.
    Reissig, J.L., Strominger, J.L., and Leloir, L. 1955. A modified colorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem. 217:959-966.
    Rondle, C.J.M. and Morgan, W.T.J. 1955. The determination of glucosamine and galactosamine. Biochem. J. 61:586-589.
    Roseman, S. and Ludowieg, J. 1954. N-acetylation of the hexosamines. J. Am. Chem. Soc. 76:301-302.
    Roseman, S. and Daffner, I. 1956. Colorimetric method for determination of glucosamine and galactosamine. Anal. Chem. 28:1743-1747.
    Tracey, M.V. 1955. A rapid colorimetric distinction between glucosamine and galactosamine. Biochim. Biophys. Acta. 17:159-160.
    Aminoff, D. 1959. The determination of free sialic acid in the presence of the bound compound. Virology 7:355-357.
    Paerels, G.B. and Schut, J. 1965. The mechanism of the periodate-thiobarbituric acid reaction of sialic acids. Biochem. J. 96:787-792.
    Powell, L.D. and Hart, G.W. 1986. Quantitation of picomole levels of sialic acid by a HPLC-adaptation of the thiobarbituric acid assay. Anal. Biochem. 157:179-185.
    Schauer, R. 1978. Characterization of sialic acids. Methods Enzymol. 50:64-89.
    Varki, A. and Kornfeld, S. 1980. An autosomal dominant gene regulates the extent of 9-O-acetylation of murine erythrocyte sialic acids: A probable explanation for the variation in capacity to activate the alternate complement pathway. J. Exp. Med. 152:532-544.
    Warren, L. 1959. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234:1971-1975.
    Hara, S., Yamaguchi, M., Nakamura, M., and Okhura, Y. 1985a. Fluorescent products of reaction between -keto acids and 1,2-diamino-4,5-dimethoxybenzene. Chem. Pharm. Bull. 33:3493-3498.
    Hara, S., Takemori, Y., Yamaguchi, M., Nakamura, M., and Okhura, Y. 1985b. Determination of -keto acids in serum and urine by high-performance liquid chromatography with fluorescence detection. J. Chromatogr. 344:33-39.
    Hara, S., Yamaguchi, M., Takemori, Y., and Nakamura, M. 1986. Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection. J. Chromatogr. 377:111-119.
    Hara, S., Takemori, Y., Yamaguchi, M., Nakamura, M., and Okhura, Y. 1987a. Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids. Anal. Biochem. 164:138-145.
    Hara, S., Yamaguchi, M., Takemori, Y., Nakamura, M., and Okhura, Y. 1987b. 1,2-diamino-4,5-methylendioxybenzene as a highly sensitive fluorogenic reagent for -keto-acids. Chem. Pharm. Bull. 35:687-692.
    Hara, S., Yamaguchi, M., Takemori, Y., Furuhata, K., Ogura, H., and Nakamura, M. 1989. Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography. Anal. Biochem. 179:162-166.
    Manzi, A.E., Diaz, S., and Varki, A. 1990. High pressure liquid chromatography of sialic acids on a pellicular resin anion-exchange column with pulsed amperometric detection: A comparison with six other systems. Anal. Biochem. 188:20-32.
    Spiker, J.E. and Towne, J.C. 1962. Fluorometric microdetermination of -keto acids. Anal. Chem. 34:1468-1471.
 Key References
    Hara et al. 1989. See above.

Describes the use of the DMB method for the detection and quantitation of Neu5Ac, Neu5Gc, and mono-O-acetylated derivatives of Neu5Ac.

    Manzi et al. 1990. See above.

Describes the application of the DMB method to other members of the sialic acid family; discusses differences in reactivity with the fluorophore and in response factors between different DMB adducts.

    Waffenschmidt and Jaenicke, 1987. See above.

Describes the modification of the original method for improved sensitivity that is used in this protocol.

    Reissig et al., 1955. See above.
    Rondle and Morgan, 1955. See above.
    Roseman and Ludowieg, 1954. See above.

Descriptions of the different modifications to the original Elson-Morgan and Morgan-Elson reactions.

    Aminoff, D. 1959. See above.
    Warren, L. 1959. See above.

Original descriptions of the TBA assay; outline factors affecting its validity and reproducibility, including reaction times, reagent concentrations, and interfering compounds.

     
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Posted on May 28, 2010 - 1:42pm
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