Determination of Galacturonic Acid Content of Pectin Using a Microtiter Plate Assay

The amount of galacturonic acid residues in samples containing pectin is an important parameter in the quantitative and structural analysis of this complex polysaccharide. This paper describes a method to determine the content of galacturonic acids in samples containing pectin, using a glass microtiter plate and microtiter plate-reading equipment with standard interference filters. The assay is a modification of a procedure involving the hydrolysis of pectin in 80% sulfuric acid at 80 °C followed by a coloring step with 3,5 dimethylphenol reagent at room temperature. The previous assay was difficult to apply routinely if large numbers of samples were to be analyzed due to color changes in the assay that are time dependent. In addition the assay involves transferring of strongly acidic solutions to a cuvette prior to reading. The use of a microtiter plate assay has several practical advantages such as an accurate estimate of background absorbance by multiple reading of the plates, and many samples can be rapidly assayed in one plate to minimize errors due to fading of the chromophore. This method is particularly advantageous when a large number of pectin samples must be analyzed for their content of galacturonic acid residues and it minimizes the transfer of strongly acidic solutions. Pectin is a structural polysaccharide commonly found in the form of protopectin in plant cells. The backbone of pectin comprises in part of 1-4 linked galacturonic acid residues. An accurate method to assay for the content of galacturonic acid is important for the quantitative and structural analysis of these complex polysaccharides. Initial work was done (Dische, 1947, 1950) using a two-step heating procedure for hydrolysis and color development. The use of hot sulfuric acid in two steps resulted in brown products or “browning” by side reactions with neutral sugars. This procedure was later was modified (Blumenkrantz and Asboe-Hansen, 1973) using mhydroxydiphenyl in the second stage of color development which reacted with uronic acids at room temperature. The room temperature step minimized browning, but browning still occurred in the first heating stage to some extent and had to be corrected for if neutral sugars were present during the assay. Another procedure was developed (Scott, 1979) using 3,5 dimethylphenol (DMP) which was claimed to be more specific for uronic acids by forming a chromophore within 10 min with a maximum absorbance of 450 nm. It was shown that neutral sugars reacting with DMP did not form a similar chromophore in the presence of chloride ion and corrections for browning could be made by subtracting the absorbance read at 400 nm. All of these procedures were typically done using a spectrophotometer equipped with standard 1 cm cuvettes and single readings were done sequentially using these instruments. Neutral and uronic acid monosaccharides can be determined by ion-exchange chromatography using dilute sodium hydroxide and sodium hydroxide-sodium acetate solutions as eluants (Clarke et al., 1991; Lee, 1990) with a pulsed amperometric detector. Although these procedures are selective, they are difficult to perform on large numbers of samples as a typical chromatogram together with re-equilibration could take up to 90 min per sample to perform. Spectrophotometers, that can read microtiter plates with 96 wells or greater, are available. The advantage of these microtiter plate spectrophotometers is that a large number of samples can be done simultaneously and the handling and transfer of hot, concentrated sulfuric acid is minimized relative to what is done using single-read spectrophotometers with 1 cm cuvettes. In addition the chromophore formed from reactions of phenolic compounds with uronic acids and neutral sugars may be unstable with a subsequent change in absorbance over time periods such as several minutes between readings. It is advantageous to take readings simultaneously in these reactions to minimize unwanted color development or bleaching that may occur between sample readings on conventional spectrophotometers, and that can introduce errors in the final reading. A microtiter plate assay for uronic acids had been developed (van den Hoogen et al., 1998) to measure hylauronic acid in synovial fluid, but a more specific microtiter plate method for galacturonic acid in pectin based on the conditions using 3,5 dimethylphenol (Scott, 1979) could be more accurate for assaying crude pectin fractions taken from plant tissue. Other microtiter procedures have been performed for hylauronic acid with carbazole but galacturonic acid had not been studied in this assay (Cesaretti et al., 2003). Material and Methods Glucose browning reaction before addition of DMP. The determination was performed in triplicate. To separate test tubes 0, 10, 25 and 50 μL of a 1% glucose (D-(+)-Glucose, SigmaUltra G7528-250G, Sigma-Aldrich, Inc., St. Louis, Mo.) solution was added. Deionized (DI) water was also added to bring the total volume up to 700 μL in each tube. Three mL of concentrated sulfuric acid (96.2% Baker Analyzed 9681-33, J.T. Baker, Inc., Phillipsburg, N.J.) containing 0.1% NaCl (S-9625 Sigma-Aldrich) was added to each tube individually and immediately vortexed for 15 sec (Vortex Genie 2—Model G-560, Scientific Industries, Inc., Bohemia, N.Y.). Each tube was then immediately placed on ice before transferring to solution basins. From each solution basin 240 μL was pipetted (12 Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Agriculture. 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