Enzymatic Assay of Pectinase
3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2 Unit Definition = = One unit will liberate 1.0 micromole of galacturonic acid from poly- galacturonic acid per hour at pH 4.0 at 25ºC.
Polygalacturonic Acid + H2O Pectinase > Galacturonic Acid
Polygalacturonic Acid + I2 > Oxidation products
2Na2S2O3+ I2 > 2NaI + Na2S4O6
The excess Iodine is titrated with Sodium Thiosulfate.
T = 25°C, pH = 4.0
Titrimetric Stop Reaction
7.3.1 0.5%(w/v) Polygalacturonic Acid (Sub)
18.104.22.168 Prepare by adding 200 mls of purified water to 1.00 g to 1.05 g of Polygalcturonic Acid, Sigma-Aldrich Product Number P3889 with stirring.
22.214.171.124 With stirring, bring to a boil and maintain boiling for exactly five minutes. Immediately place in a 25ºC water bath with an immersible stirrer. Stir until the temperature is at 25ºC and allow the solution to stir for an additional ten minutes.
126.96.36.199 With continued stirring, adjust the pH of the solution by adding 0.05 ml aliquots of Reagent 7.3.6 (NaOH-2) at 1 minute intervals until a pH of 4.0 is obtained. Add 0.05 ml aliquots of 2 N NaOH with stirring until a pH of 4.00 to 4.05 is maintained for a minimum of ten minutes. If particles are present, then filter through an Evergreen Column, maximum 90 μm frit.
188.8.131.52 Recheck pH and adjust with Reagent 7.3.3 (NaOH) at 25ºC to 4.00 - 4.02 with stirring. The pH should remain in the range of 4.00 - 4.02 for a minimum of 30 minutes.
184.108.40.206 It may be necessary to recheck pH every fifteen minutes to ensure that the pH is within the range of 4.00 to 4.02 at 25ºC. If not, adjust pH with 0.1N NaOH.
7.3.2 100 mM Iodine (I2)
Use Iodine volumetric Standard, 0.1 N, Sigma-Aldrich Product Number 318981.
7.3.3 1.0 N Sodium Hydroxide (NaOH)
Use 1.0 N Sodium Hydroxide Solution, Sigma-Aldrich Product Number S2567.
7.3.4 1 M Sodium Carbonate (Na2CO3)
Prepare in purifed water at 106 mgs/ml using Sodium Carbonate, Reagent Grade, Sigma-Aldrich Product Number S2127.
7.3.5 2.0 N Sulfuric Acid (H2SO4)
Prepare in purified water at 0.06 ml/ml using Sulfuric Acid, ACS Reagent, Sigma-Aldrich Product Number 258105.
7.3.6 10 N Sodium Hydroxide (NaOH-2)
Prepare at 400 mgs/ml in purified water using Sodium Hydroxide, Reagent Grade, Sigma-Aldrich Product Number S5881.
7.3.7 100 mM Sodium Thiosulfate, Standardized (Na2S2O3)
Prepare in 3 liters of purifed water using 78.3 g of Sodium Thiosulfate, Pentahydrate, Sigma-Aldrich Product Number S8503 and 0.6 g of Sodium Carbonate, Monohydrate, Sigma-Aldrich Product Number S4132. Standardize against a standard solution of potassium dichromate, prepared from potassium dichromate, NIST.
7.3.8 Pectinase Solution (Enzyme)
Immediately before use, prepare a solution containing approximately 100 unit/ml in cold purified water. Dissolution may require more than one minute of swirling and some insoluble particulates may still be present.
7.3.9 1.0 %(w/v) Starch (Starch)
Prepare in purified water at 10 mgs/ml by boiling for exactly five minutes with stirring. Cool to room temperature. Use Starch, Potato Soluble, Sigma-Aldrich Product Number S2004.
7.4 TEST METHOD
7.4.1 Pipette (in milliliters) the following reagents into suitable glass vessels in triplicate for control and/or sample:
|Reagent 7.3.1 (Sub)||4.90||4.90|
7.4.2 Mix by swirling and equilibrate to 25ºC for a minimum of three minutes. Then add:
|Reagent 7.3.8 (Enzyme)||0.100||------|
7.4.3 Mix by swirling and incubate Test and Blank for exactly 5 minutes at 25ºC. Then add the following:
|Reagent 7.3.2 (I2)||5.00||5.00|
|Reagent 7.3.4 (Na2CO3)||1.00||1.00|
7.4.4 Mix by swirling and place in the dark for exactly 20.0 minutes. Then add:
|Reagent 7.3.5 (H2SO4)||2.00||2.00|
7.4.5 Mix by swirling and place on a stirrer at room temperature. Titrate with Reagent 7.3.7 (Na2S2O3) until the solution is a faint yellow color. Then add the following:
|Reagent 7.3.9 (Starch)||0.10||0.10|
7.4.6 With stirring, continue titrating with Reagent 7.3.7 (Na2S2O3) until the solution is colorless. Record the amount in milliliters.
|7.5.1||Units/ml enzyme =||(mls of titrant for blank - mls of titrant for test)(1)(df)(100)|
df = Dilution Factor
1 = One micromole of galacturonic acid is oxidized by 1 micrroequivalent of I2
100 = Microequivalents of S2O3 per milliliter of titrant
0.100 = Volume (in milliliter) of enzyme used in enzymatic reaction
2 = Microequvalents of S2O3 oxidized per microequivalent of I2 reduced
5.0 = Time of incubation of assay in minutes per unit definition
|7.5.2||Units/mg protein =||Units/ml enzyme|
|mg protein/ml enzyme|
7.6 FINAL ASSAY CONCENTRATION
In a 5.0 mL reaction mix, the final concentrations are 0.49%(w/v) Polygalacturonic Acid and 10 units of Pectinase.
8.1 Kertesz, Z. I. (1955) Methods in Enzymology. 1, 162-164.
8.2 Replaces OP PEPOLY02.
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