Plant Cell Wall Degrading Enzymes Assays
Polygalacturonase
1. Prepare a 1 liter solution of 2.5 g Polygalacturonate (grade II, Sigma), in 0.1 M sodium acetate pH 6.
2. Add enzyme preparation.
3. Remove 50 ul samples and add to 1 ml of 0.5 M Na2CO3.
4. Use neocuproin reagent to determine the amount of reducing sugars.
1 unit of enzyme = 1 umol of reducing sugar released in 1 hour.
(from Hugouvieux-Cotte-Pattat et al. 2002. J. Bacteriol 184:2664-2673.)
A232 Assay for Pectate Lyase Activity
1. Prepare 200 ml of substrate for the final reaction mixture (0.1% PGA, 100 mM Tris-HCl pH 8.5, and 1.5 mM CaCl2)
a. Dissolve 210 mg of PGA in 100 ml of 210 mM Tris-HCl pH 8.5. After adding the PGA, adjust pH to 8.5 with NaOH if necessary.
b. With a stir bar swirling vigorously, add (slowly, against the wall of the beaker) 100 ml of 3.0 mM CaCl2. Inspect for any pectate gel. If gel is present, discard the solution and start over with better mixing and add the calcium solution more slowly.
c. Do not add sodium azide as this interferes with the assay. Instead, filter sterilize the substrate mix and store at 4C. Aliquot substrate as needed.
Notes on PGA. Polygalacturonase acid preparations vary in chain length and purity. Unfortunately, the Sunkist brand preps were best for this assay and it is no longer available. Several brands may need to be tried to find a brand that works.
A232 Assay
1. Warm substrate to assay temperature (typically room temperature)
2. Put 1.9 ml of substrate in a 3-ml quartz cuvette with a 1 cm light path.
3. Add 0.1 ml of enzyme sample plus water (previously equilibrated to assay temperature) and mix rapidly using parafilm to seal the top of the cuvette. For example, if only 20 ul of enzyme is added, then use 80 ul of water.
4. Monitor the increase in A232 over time. The increase should be linear and the r value should be >0.9990. For samples with very high activity, linearity may be achieved by shorter assay time periods or higher dilution of the enzyme.
Calculating units of activity (umol unsaturated product formed / minute / ml of enzyme sample)
1. The molar extinction coefficient for the unsaturated product at 232 nm is 4600 M-1 cm -1.
2. Hence, in a 2 ml assay, an A232 reading of 1.0 = 0.433 umol. Therefore, if 100 ul of enzyme caused and absorbance increase of 1.0 in 5 min., the reaction rate reading will be 0.20 A232/min and the activity of the sample = 0.433 umol/A232 x 1000 ul/100 ul x 0.2A232/min = 0.87 umol/min.
Important points:
a. The objective is to determine the capacity of 1 ml of enzyme sample to generate unsaturated uronide. THe value of 0.433 umol/A232 already contains a correction for the 2 ml reaction volume in the cuvette. Hence, if you use 100 ul of your sample, multiple the results by 10 and not by 20.
b. Therefore, when the spec asks for a "factor," which is used to calculate the "result," enter {(0.433 x 1000) / (ul of enzyme used in the assay)}. The result will then be in Pel units of umol unsaturated product formed/min/ml of enzyme sample.
(Protocol obtained from Collmer Lab)