II. Cellulose Determination; Total Sugar and Uronic Acids
III. Fractionation of Cell Wall Polysaccharides
IV. Methyl Esterification of Uronic Acids
VI. Linkage (methylation) Analysis
VII. Fourier Transform Infrared (FTIR)
II. Cellulose Determination; Total Sugar and Uronic Acids
A. Acid Hydrolysis
1. Use 1 mg cell wall in a 0.75ml conical vial. In a fume hood, add 1 mL
of 2 M TFA with 1µmole myo-inositol and cap tightly with cap lined with
Teflon.
2. Heat at 120°C for 90 min with vortex every 30 min. to break up any
chunks. Make sure that the cap hasn't loosened during heating or you'll launch
the tube across the fume hood.
3. Centrifuge the suspension at 2,500rpm for 2 minutes.
4. Remove supernatant and continue to step 5. Reserve pellet for cellulose determination.
5. Non-cellulose monosaccharide in supernatant liquid under stream of air at 40-45°C.
6. Use pellet from part A, step 4.
7 . Wash pellet 5 times with water.
8. Centrifuge to remove all Water.
9. Resuspend in a known volume (depending on amount of pellet).
10. Take a portion of the resuspended pellet and add to a test tube; add
11. Use phenol-sulfuric assay to determinate glucose equivalents.
Standards: 0.005 to 0.05 mg microcrystallines cellulose /100 µL of H2O
B. Phenol-sulfuric assay for total sugar.
Adapted from the method of Dubois et al. (1956), which should still be the citation of choice. This method is exceptionally useful to monitor column fractions. The inherent problem with the method is that different monosaccharides react differently and give varying amounts of chromagen of different absorbance maxima. However, if you know the know the general composition, then you are informed as to the standards to run. Because the method employs concentrated H2SO4, then even cellulose is hydrolyzed, and, thus, the method can be used in assay of cellulose and other similar para-crystalline polysaccharide aggregates.
Mix in a small (6-ml) glass tube:
100 µL sample in water or 50 mM Na acetate
100 µL freshly made 5% phenol in water
Briskly add 1 ml of conc. H2SO4 into the mixture.
Immediately vortex-mix several seconds. An orange color with intensity proportional concentration will begin to develop immediately and reach a maximum about 2 h at 30°C. Read against standards at 500 nm. Sensitive in a range of about 0.01 to 0.15 mg of cellulose/100 µL, or glucose equivalent of 10 to 300 nmoles.
Dubois M, Gilles DA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28, 350-356.
C. Determination of Uronic Acids
This procedure is a modification of that of Galambos [Galambos, J. T. (1967) Anal. Biochem. 19, 119-132] and Blumenkrantz & Asboe-Hansen [Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54, 484-489] who introduced sulfamate and the diphenyl reagent, respectively, for reduction of interference by neutral sugar. In old methods using carbazole, the uronic acids gave a pretty pink color whereas neutral sugars in 10-fold excess gave an ugly brown color that interfered with detection with the uronic acid. Combination of the sulfamate to depress browning and diphenyl to color the uronics at ambient temperaure (Filisetti-Cozzi & Carpita. 1991. Analyt. Biochem 197, 157-162 ) substantially reduces this browning due to neutral sugars. The color is not quite pretty pink in excess neutral sugar but rather pink-amber to orange depending on the kind and amount of neutral sugar. Absorbance at 512 nm is nevertheless close to standard values. Color production by uronics is reduced somewhat in the presence of interfering neutral sugars, so it might be necessary to scan the chromagen from 400 to 700 nm and "manually" subtract the background absorbance between 630 and 450 nm (i.e. you print the scan, cut out the chromagen peak minus background and weigh it)
Reagents:
4 M sulfamic acid/potassium sulfamate:
MW= 97.09 = 38.84 g per 100 mL
Suspend crystals in 50 mL of water by vigorous stirring and added concentrated
KOH until sulfamic acid just dissolved. Cool to room temperature and pH to
1.6 with additional KOH. Bring to volume and store in glass scintillation
vials.
0.15% m-hydroxydiphenyl in 0.5% NaOH:
Dissolve 15 mg of diphenyl in 10 mL of fresh 0.5% NaOH (use 1:99 dilution
of 50% low-carbonate NaOH) and store in dark bottle (or foil-wrapped bottle).
?Keep refrigerated but make fresh before use
--or--
0.1% carbazole in EtOH (1 mg/ml)
20 mM D-galacturonic (or D-glucuronic) acid standard
Assay:
1. Dry samples of cell wall or cell-wall fractions are suspended in water
to 0.5-1mg/ml to contain up to 200 nmol of uronic acid per 0.4 mL of solution.
If the suspension does not dissolve, then sonicate and stir intermittently
until the big chunks break uip. Cut off the tip of 1 mL Rainin pipet tip and
dispense 0.4 mL of the even suspension into a 6-mL tube. Do at least in duplicate.
2. Add exactly 40 µL of 4 M sulfamic/K-sulfamate to the 0.4 mL of sample
and mix in by vortex.
3. Add 2.4 mL of concentrated H2SO4 or (H2SO4 + 75 mM sodium tetraborate)
by big pipet. Inject the stream directly into the 0.4 mL solution at the bottom
of the tube, if possible, and vortex mix up-and-down until well mixed.
4. We found that sulfamate suppresses neutral sugar interference such that
carbazole is a better reagent than the m-hydroxybiphenyl. Add 100 ?L of the
carbazole reagent. Vortex mix very thoroughly.
4. Marble tubes and place in boiling water bath for 20 min. Remove rack of
tubes and plunge into ice-water until cooled to room temperature.
--or, if you insist…--
5. Skip carbazole additions before boiling, and instead, add 80 µL of the m-hydroxybiphenyl reagent after cooling and vortex mix. Let color develop for at least 10 min and read at 525 nm. Color is unstable, so read in a timely manner.
If there is still substantial sugar interference, run standard curve of 200 nmol of appropriate uronic acid in 10 or 20-fold excess neutral sugar. Then scan the chromagen from 400 to 700 nm and "manually" subtract the background absorbance between 630 and 450 nm.
Stock standards are diluted with water 1:19 to give 1 mM standard curve stocks. Run your curve from 50 to 300 nmol per 0.4 mL of sample volume (i.e 50 to 300 µL of 1 mM stock and 350 to 100 µL of water balance).
Use neutral sugar interference stock (20 mM) without dilution to give 10
to 20-fold excess (as necessary). For example 200 µL of 1 mM uronic acid stock
and 200 µL 20 mM sugar will give 200 nmol of uronic acid and 4000 nmol of
sugar in a total volume of 0.4 mL.
Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54,
484-489.
Galambos, J. T. (1967) Anal. Biochem. 19, 119-132.
Filisetti-Cozzi, T. M. C. C., and Carpita, N. C. (1991) Anal. Biochem. 197,
157-162.
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