Determination of the Uronic Acid Content of Plant Cell Walls Using a Colorimetric Assay

Uronic acid in the form of galacturonic acid is a major component of the pectic polysaccharide rhamnogalacturonan, which is present in large amounts in the cell walls of most fruits and vegetables. Small amounts of glucuronic acid and 4-O-methylglucuronic acid have also been detected in cell walls. Quantitative measurement of total uronic acid is commonly done using colorimetric methods after first hydrolyzing the polysaccharides in sulfuric acid (Ahmed and Labavitch, 1977; Selvendran et al., 1979). However, there is a major problem with the older methods of determining uronic acid content of cell walls. Neutral sugars and their degradation products from acid hydrolysis can interfere in the colorimetric determination of uronic acids. The procedure developed by Filisetti-Cozzi and Carpita (1991) solves the problem, allowing uronic acids to be determined in up to ten times their weight of neutral sugars. For the determination of neutral sugars see UNIT E3.2. The procedure can be used to determine mannuronic and iduronic acids. With glucuronic acid as the standard, the protocol could be used to measure the glucuronic acid contents of xanthan and gellan gums. Cell walls and cell wall fractions are first hydrolyzed using the method of Ahmed and Labavitch (1977) and the uronic acid content estimated using the method of Filisetti-Cozzi and Carpita (1991). Materials Dried cell walls or cell wall fractions (see UNIT E3.1) Concentrated sulfuric acid 4 M sulfamic acid/potassium sulfamate solution, pH 1.6 (see recipe) 75 mM sodium tetraborate (see recipe) m-hydroxydiphenyl solution (see recipe) 0.5% (w/v) sodium hydroxide (see recipe) D-galacturonic acid standards (see recipe) Borosilicate glass tubes and caps 10-ml volumetric flasks 15-ml centrifuge tubes 15-ml borosilicate glass tubes 100C water bath Spectrophotometer CAUTION: The uronic acid assay involves the use of concentrated sulfuric acid. Wear goggles, gloves, and protective clothing. Hydrolyze cell walls or cell wall fractions 1. Weigh 5 mg (record exact weight) of cell walls or cell-wall fraction (in duplicate) into borosilicate glass tubes (see UNIT E3.1 for cell wall preparation and cell-wall fractionation). 2. Add 1 ml concentrated sulfuric acid to all the tubes and cap. Set up a reagent control tube containing only 1 ml concentrated sulfuric acid and carry this through all the procedures.

UNIT E3.3

BASIC PROTOCOL

Cell Wall Polysaccharides Contributed by Laurence D. Melton and Bronwen G. Smith

Current Protocols in Food Analytical Chemistry (2001) E3.3.1-E3.3.4 Copyright 2001 by John Wiley & Sons, Inc.

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3. Place the tubes in an ice bath and stir the contents for 5 min. Do this by placing small spin bars in each of the tubes and placing the rack of tubes in a container of ice slurry on a magnetic stirrer (take care not to splatter the solid material above the level of the liquid). 4. Add another 1 ml concentrated sulfuric acid to all the tubes and stir on ice for 5 min (take care not to splatter the solid material above the level of the liquid). 5. Add 0.5 ml water and stir for 5 min on ice. 6. Add another 0.5 ml water and stir for 5 min on ice. 7. Dilute the contents of each tube with water to 10 ml in a 10-ml volumetric flask. Transfer to a 15-ml centrifuge tube and centrifuge for 10 min at 2000 g, room temperature, to pellet any unhydrolyzed material. Perform colorimetric assay 8. For each hydrolysate, set up three 15-ml borosilicate glass tubes. For the reagent control, set up two tubes. Take aliquots of 400 l from each hydrolysate supernatant and reagent control and place in the respective tubes. 9. Add 40 l of 4 M sulfamic acid/potassium sulfamate solution, pH 1.6, to all the tubes. Vortex contents of the tubes. 10. Add 2.4 ml of 75 mM sodium tetraborate in sulfuric acid solution to all the tubes. Vortex vigorously. 11. Place the tubes in a 100C water bath (boiling) for 20 min, then cool by plunging tubes into an ice bath for 10 min.
Place glass marbles on top of the tubes to prevent contamination of the samples by condensation.

12. Add 80 l m-hydroxydiphenyl solution to 2 tubes of each sample and the 2 reagent control tubes. Add to the third tube of each sample 80 l of 0.5% NaOH (this is the sample control). Vortex the contents of the tubes three times; ensure they are mixed well.
A pink color develops within 5 to 10 min and is stable for 1 hr after which it fades.

13. Between 10 min and 1 hr after complete mixture, read the absorbances at 525 nm against the reagent control. Subtract the values for the sample controls from their corresponding sample absorbances. 14. Prepare a D-galacturonic acid standard curve with each batch of samples. REAGENTS AND SOLUTIONS
Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
D-Galacturonic

acid standards Thoroughly dry D-galacturonic acid over a desiccant (e.g., phosphorus pentoxide). Make up a stock standard solution and generate a standard curve from this. Prepare a stock solution of 20 mg/ml of galacturonic acid by weighing 20 mg of the dried D-galacturonic acid into a vial and adding 1 ml of water to dissolve (this stock solution can be stored frozen). Then prepare a 200 g/ml solution by taking 100 l of the stock solution and adding 9900 l of water. Use this solution to prepare the dilution series in Table E3.3.1.
CAUTION: Phosphorus pentoxide dessicant is corrosive; wear goggles, gloves, and protective clothing.
continued

Colorimetric Assay of Uronic Acid Content of Cell Walls

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Table E3.3.1

Dilution Series for the D-Galacturonic Acid Standard

Concentration of galacturonic acid (g/400 l) 5.0 10.0 15.0 20.0 30.0 40.0

Volume of 200 g/ml galacturonic acid solution (l) 125 250 375 500 750 1000

Volume of water (l) 1875 1750 1625 1500 1250 1000

It may be necessary to prepare an alternative dilution series depending on the uronic acid content of your samples. Store the stock solution frozen and prepare fresh dilutions from this for each batch.

m-Hydroxydiphenyl solution Weigh out 0.15 g of 3-phenylphenol into a 100-ml volumetric flask, dissolve in <100 ml of 0.5% (w/v) sodium hydroxide (see recipe) then adjust the final volume to 100 ml with 0.5% sodium hydroxide. Store in a dark bottle (or wrap the bottle in aluminum foil) at 4C. The solution is stable for 1 month. Sodium hydroxide, 5% (w/v) Place 0.5 g of NaOH into a 100-ml volumetric flask. Add 20 ml water to dissolve the pellets and then adjust the volume to 100 ml with water. Prepared fresh. Sodium tetraborate solution, 75 mM To prepare 100 ml of 75 mM solution, weigh out 1.501 g of sodium tetraborate (mol. wt., 201.2) into a 100-ml volumetric flask and add 90 ml concentrated sulfuric acid, and place a stopper in the flask. Stir until dissolved then adjust the final volume to 100 ml with sulfuric acid.
Ensure the sodium tetraborate is completely dissolved in the sulfuric acid. This usually requires stirring overnight. For preparation wear goggles, gloves, and protective clothing. Prepare only sufficient amount for immediate use. Keep at room temperature.

Sulfamic acid/potassium sulfamate solution, 4 M (pH 1.6) Weigh out 38.84 g of sulfamic acid (mol. wt., 97.09) and stir vigorously in 50 ml of water. Add saturated KOH dropwise until the sulfamic acid has dissolved. Allow the sulfamic acid solution to cool and then carefully adjust the pH to 1.6 with saturated KOH. Adjust the volume to 100 ml with water to give a final concentration of 4 M. Store at room temperature. COMMENTARY Background Information
Uronic acids were traditionally measured by the carbazole reaction. In this procedure, sugars were treated with concentrated sulfuric or hydrochloric acid to yield products that reacted with carbazole. For example, Dische (1947) used carbazole for the determination of uronic acid in solutions of sulfuric acid, but the method was subject to interference from neutral sugars that were also present in the solution. The method was subsequently modified by Gregory (1960) and Bitter and Muir (1962) by the inclusion of borate in the sulfuric acid mixture together with a heating step. This procedure gave an increased color production but did not overcome the problem of interference by neutral sugars. A further modification was made by Galambos (1967) in which sulfamate was incorporated into the mixture prior to the heating step. Addition of sulfamate overcame the

Cell Wall Polysaccharides

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interference problem, but the sulfamate was inclined to precipitate out; therefore, further modifications were required to deal with this. A slightly different approach was made by Blumenkrantz and Asboe-Hansen (1973). They still had sodium tetraborate in sulfuric acid, but had m-hydroxydiphenyl as the color reagent instead of carbazole. Unfortunately, this method was also susceptible to interference from neutral sugars. A major breakthrough in the determination of uronic acid was made by Filisetti-Cozzi and Carpita (1991) who retained the use of sodium tetraborate in sulfuric acid and m-hydroxydiphenyl as the color reagent but incorporated sulfamate into the mixture. The Filisetti-Cozzi and Carpita (1991) method is now the colorimetric method of choice because it can determine uronic acids in the presence of up to 10 times the weight of neutral sugars. The sulfamate suppresses the formation of brown pigments from the neutral sugars and tetraborate increases the sensitivity of the reaction with uronic acids.

of replicate samples should be within 0.005 units.

Time Considerations
Allow a few days to prepare for this assay. The D-galacturonic acid standard has to be dried over a desiccant for several days and the sodium tetraborate requires stirring overnight to dissolve in sulfuric acid. The time to complete this assay will depend on the number of samples one does in a batch. For the first attempt, it is suggested that only two samples be analyzed along with the standard curve. This will probably take 4 hr. Although it is tempting to do large numbers in a batch, it does take time to read them all on the spectrophotometer, especially if only read one at a time, and within the hour.

Literature Cited
Ahmed, A.E.R. and Labavitch, J.M. 1977. A simplified method for accurate determination of cell wall uronide content. J. Food Biochem. 1:361365. Bitter, T. and Muir, H.M. 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4:330334. Blumenkrantz N. and Asboe-Hansen G. 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54:484-489. Dische, Z. 1947. A new specific color reaction of hexuronic acids. J. Biol. Chem. 167:189-198. Filisetti-Cozzi, T.M.C.C. and Carpita, N.C. 1991. Measurement of uronic acids without interference from neutral sugars. Anal. Biochem. 197:157-162. Galambos, J.T. 1967. The reaction of carbazole with carbohydrates. I. Effect of borate and sulfamate on the carbazole color of sugars. Anal. Biochem. 19:119-132. Gregory, J.D. 1960. The effect of borate on the carbazole reaction. Arch. Biochem. Biophys. 89:157-159. Selvendran, R.R., March, J.F., and Ring, S.G. 1979. Determination of aldoses and uronic acid contents of vegetable fiber. Anal. Biochem. 96:282292.

Critical Parameters
Accurate pipetting of small volumes is essential for reproducibility. The absorbances should be read within 1 hr; the color fades beyond this time.

Troubleshooting
Take care to scrupulously clean glassware, as lint and other material will be degraded by concentrated sulfuric acid. Ensure that there are no bubbles in the cuvettes when reading the absorbances. Hold the cuvettes up to the light and check before placing them in the spectrophotometer. Ensure that there are no spillages down the sides of the cuvettes as the sulfuric acid is very corrosive and may damage equipment. If a large excess of neutral sugars is present or browning is observed, determine the amount of neutral sugars present (UNIT E3.2) in the sample. Record the absorbance spectrum (400 to 700 nm) of the corresponding amount of neutral sugars and subtract it from the spectrum of the colorimetric reaction. The presence of other polysaccharide gums that contain uronic acids (alginates, xanthan, and gellan) causes serious interference.

Key Reference
Filisetti-Cozzi and Carpita, 1991. See above. An excellent account of method development.

Anticipated Results
Colorimetric Assay of Uronic Acid Content of Cell Walls

Careful technique should result in reliable results with high reproducibility. Absorbances

Contributed by Laurence D. Melton and Bronwen G. Smith University of Auckland Auckland, New Zealand

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