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.
Pectin
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.
REAGENTS:
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.
PREPARATION AND MEASUREMENT OF SAMPLES:
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.