The unit of the unsaturated product formed is Molar (M).
The unsaturated digalacturonide is measured at 235 nm and assumes the molar extinction coefficient of 4600 (M-1 cm-1). Pectate lyase liberates various unsaturated oligogalacturonides and therefore this test is qualitative rather than quantitative.
Assay using m-Hydroxydiphenyl
Galacturonic acid residues form the fundamental units of pectin molecules. A quantitative measurement of this acid is used to determine the concentration of pectic substances present in a sample. The colourimetric assay using m-hydroxydiphenyl for analysis of galacturonic acids is quite specific for uronic acids. It can tolerate, for instance, the presence of up to 1000 ppm of sucrose.
PREPARATION AND MEASUREMENT OF SAMPLES:
1. Galacturonic acid stock solution:
Dissolve 100 mg dry galacturonic acid powder in 100 mL distilled water to give a solution concentration of 1 mg/mL. Keep refrigerated. A new solution should be prepared every 4 weeks.
2. M/80 Sodium tetraborate in sulphuric acid (0.0125M solution):
Weigh 1.192 g of sodium tetraborate (Na2 BO4.10H20) into a 250 mL volumetric flask. Make up to the mark with concentrated sulphuric acid.
3. 0.5% sodium hydroxide:
Weigh 5 g sodium hydroxide into a 1 litre volumetric flask. Make up to the mark with deionised water.
4. m-Hydroxydiphenyl solution (0.15%):
Weigh 0.15 g m-hydroxydiphenyl into a 100 mL volumetric flask. Make up to the mark with 0.5% sodium hydroxide solution. Cover the container with aluminium foil to protect it from the light. Keep refrigerated.
Make the solutions for the calibration curve as follows:
Pipette 2 mL stock galacturonic acid solution into a 100 mL volumetric flask. Make up to the mark with deionised water. The concentration of galacturonic acid in this sample is equivalent to 20 micrograms/mL.
4 mL stock in 100 mL volumetric flask for 40 micrograms/mL
6 mL stock in 100 mL volumetric flask 60 micrograms/mL
8 mL stock in 100 mL volumetric flask 80 micrograms/mL
10 mL stock in 100 mL volumetric flask 100 micrograms/mL
Place 16 test tubes in an ice bath to cool. Use three tubes for each sample: two tubes for sample + one tube for blank determination.
Keep the sulphuric acid/sodium tetraborate solution in an ice bath throughout the experiment.
Place 1.0 mL of standard or sample into each of three labelled cold test tubes. Allow a few minutes to cool.
Add 6.0 mL of the tetraborate solution to each test tube. Mix thoroughly by means of a test-tube stirrer. It is important that the sample plus reagents are mixed properly. Keep test tubes in ice until all samples are prepared. Heat tubes in a boiling water bath for precisely 6.0 mins. Return tubes to the ice bath. Allow to cool.
Add 0.1 mL (100 microlitres) of m-hydroxydiphenyl to the first two tubes to develop colour. Mix thoroughly using the test-tube stirrrer. Add (for blank) 0.1 mL (100 microlitres) of 0.5% sodium hydroxide to the third tube. Mix thoroughly. Allow the tubes to stand for 15-20 minutes at room temperature to allow any bubbles formed to dissipate.
Measure absorbance at 520 nm on a spectrophotometer by reading sample against corresponding blank tube with 0.5% sodium hydroxide.
To obtain the absorbance due to m-hydroxydiphenyl, substract the absorbance for sample blank from the total absorbance for sample.
Zero the spectrophotometer with a reagent blank prepared by mixing 10 mL deionised water plus 6.0 mL tetraborate solution and 0.1 mL of 0.5% sodium hydroxide solution.
Plot a calibration curve of Absorbance (y-axis) against concentration of galacturonic acid (x-axis). Use this curve for standardisation.