D-Glucuronic acid and D-Galacturonic acid,

UV method
Catalogue number: AK00221, 100 tests

Application 100 L. The detection limit is 17.4 mg/L, which is derived
from an absorbance difference of 0.020 and a sample volume
This rapid and simple method is used for the determination of 100 L.
of D-glucuronic acid and D-galacturonic acid in a variety of
matrices. This kit is adequate to D-hexuronic acids (including
Linearity and precision
D-glucuronic acid and D-galacturonic acid) measurement in
hydrolysates of plant material and polysaccharides, as well as The assay is linear over the range of 5 to 150 μg of D-
other materials. glucuronic acid or D-galacturonic acid per assay. In duplicate
determinations using one sample solution, an absorbance
Introduction difference of 0.005 to 0.010 may occur. With a sample
volume of 0.100 mL, this corresponds to a D-glucuronic acid
D-Glucuronic acid and D-glucuronic acid are naturally or D-galacturonic acid concentration of approx. 4.4 to 8.7
occurring hexuronic acids present in glycosaminoglucans, mg/L of sample solution.
glucuronid conjugates in plant polysaccharides and in
mammals. Both D-glucuronic acid and D-glucuronic acid are
Interferences
major components of plant cell wall polysaccharides, being
D-glucuronic a component of arabinoxylan and D- If the conversion of D-glucuronic acid or D-galacturonic acid
galacturonic the major component of pectin. In mammals, D- has been accomplished within the time specified in the assay
glucuronic acid occur as a component of (10 min at 25ºC), it can be generally concluded that no
glycosaminoglucans, such as hyaluronan, heparin and interference has occurred. Nevertheless, this can be
chondroitin present in cartilage. confirmed by adding D-glucuronic acid or D-galacturonic
acid to the cuvette on completion of the reaction. A
Principle substantial increase in absorbance should be observed. An
internal standard should be included during sample analysis
if the presence of interfering substances is suspected. A
UDH quantitative recovery of this standard should be expected.
D-glucuronic acid + NAD+ + H2O D-glucarate + NADH + H+

UDH
Kit composition
D-galacturonic acid + NAD+ + H2O D-glalactarate+ NADH + H+
Solution 1. Buffer (22 mL, pH 8.0) plus sodium azide (0.02%
w/v) as a preservative. Stable for 2 years at 4 °C.
+
Solution 2. NAD freeze dried powder. Stable for 5 years at
The amount of NADH formed through the action of uronate
-20 °C.
dehydrogenase (UDH) measured at 340 nm, is stoichiometric
with the amount of D-glucuronic and/or D-galacturonic acid Dissolve content in 22 mL of distilled water and divide into
present in sample volume. appropriately sized aliquots and store in PP tubes at -20 °C
between use (stable for 2 years) and keep cool during use.
Specificity Suspension 3. Uronate dehydrogenase (UDH) in 3.2 M
The method is specific for D-Glucuronic acid and D- ammonium sulphate (2.2 mL). Stable for 2 years at 4 °C. Swirl
glucuronic acid. bottle before use.

Solution 4. D-glucuronic standard solution (5 mL, 0.5

Sensitivity and detection limit mg/mL). Stable for >2 years at 4 °C. This standard solution
The smallest differentiating absorbance for the assay is 0.01 can be used when there is some doubt about the method
AU. This corresponds to a hexuronic acids concentration of accuracy.
8.7 mg/L of sample solution with a sample volume of
Safety
Reagents that are used in the determination of hexuronic
acids are not hazardous materials (see Hazardous Substances
Regulations). However, the concentrated buffer contains
sodium azide as a preservative. The general safety measures
that apply to all chemical substances should be followed.
Procedure
Calculation
Determine the absorbance difference (A2-A1) for both blank
and sample. Subtract the absorbance difference of the blank
from the absorbance difference of the sample, thereby
obtaining ΔAD-hexuronic acid.
The concentration of D-hexuronic acids (D-glucuronic acid or
D-galacturonic acic) (g/L) is calculated as follows:

Wavelength: 340 nm

Cuvette: 1 cm light path (glass or plastic) C (D-hexuronic acid) = 1.033 x AD-hexuronic acid g/L

Temperature: 25 °C or 37ºC

Final volume: 2.52 mL If the sample has been diluted or a different sample volume
Sample solution: 5-150 μg of D-glucuronic acid or D- was used during the reaction, the result must be multiplied
galacturonic acid per cuvette (in 0.1 to 2.0 0l sample volume) by the corresponding dilution/concentration factor.

Read against air (without a cuvette in the light path) or against

water
2

Absorbance @ 340 nm
Pipette into cuvettes (mL) Blank Sample 1.5
Distilled water 2.10 2.00
1
Sample - 0.10
Solution 0.20 0.20 0.5
Solution 0.20 0.20
0
Mix, measure the absorbances of the above solutions
0 25 50 75 100 125 150
(A1) after approx. 3 min and start the reactions by
addition of g/test

Suspension 3 (UDH) 0.02
Mix, measure the absorbances of the above solutions
(A2) at the end of the reaction (approx. 10 min)*
Mixtures can be obtained with a plastic spatula or by gentle
inversion after sealing with a cuvette cap or Parafilm®.
* if necessary, continue to read the absorbances at 2 min
intervals. The reading should remain the same over 1 min
interval (Figure 1).
0.02 Figure 2. Standard curve relating D-glucuronic concentration
(g/test) to absorbance at 340 nm (25 ºC)
Sample preparation
Sample dilution
The amount of D-glucuronic acid or D-galacturonic acic
present in the cuvette should range between 5 and 150 μg.
Thus, the sample solution must be diluted to yield a
concentration between 0.05 and 1.5 g/L.

2 150 g Examples of sample preparation

Determination of D-glucuronic acid or D-galacturonic
125 g
Absorbance @ 340 nm

1.5 acic in cell culture medium and fermentation samples

100 g To inactivate enzyme activity, incubate an aliquot ( 10mL) of
75 g the sample solution at 90-95ºC for 10 minutes. Filter or
1
centrifuge and use clear filtrate or supernadant in the assay.
50 g As alternative, deproteinization can be achieved with Carrez
0.5 reagents. Homogenise solid agar media with water and treat
25 g as described previously. Dilute properly in distilled water and
5 g use in the assay.
0 0 g
Determination of D-glucuronic acid or D-galacturonic
0 2 4 6 8 10 12 14 16
acic in plant samples
Incubation time (min)
Pulverise plant sample to pass a 0.5 mm screen. Weight 1.0 g
Figure 1. Increase in absorbance at 340 nm on incubation of 0-150 of sample and extract with 90 mL of heated water (80ºC).
g of D-glucuronic acid, performed with NZYTech kit at 25 ºC using Quantitatively transfer to a 100 mL volumetric flask and
1 cm pathlength cuvettes. dilute to the mark with distilled water. Mix, filter and use the
clear solution in the assay. Dilute properly in distilled water filtrate or supernadant in the assay. Dilute properly in
and use in the assay. distilled water and use in the assay.
Determination of D-glucuronic acid or D-galacturonic
acic in polysaccharides and fibrous plant samples References

Pulverise plant sample to pass a 0.5 mm screen, using a Moon T.S., Yoon S.H., Ching, M.J., Lanza A. and Prather K. L. J
centrifugal mill. Accurately weight 100 mg of material into a (2009). Enzymatic assay of D-glucuronate using urinate
screw-cap culture tube. Add 5 mL of 2M H2SO4, cap the tube dehydrogenase.. Anal. Chem., 392, 183.185.
and incubate at 100ºC for 6 h stirring intermittently. Cool the
tube to room temperature, carefully loosen the cap and add
7 mL of 2 M NaOH. Quantitatively transfer to a 100 mL Released 11/15
volumetric flask and dilute to the mark with distilled water.
Mix thoroughly by inversion. Filter or centrifuge and use clear

Certificate of Analysis

Test Criteria Result

Test Performance Reaction completed within time stated Meets specification

Target value for recommended standard material +/- 10% Meets specification

Blank reaction absorbance +/- 10% of the blank value Meets specification

Approved by:
José Prates
Senior Manager, Quality Systems

Please enquire info@nzytech.com to obtain any additional information about this kit,

including additional specific applications.

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