Enzymatic Assay of Pectinase

1. Objective

To standardize a procedure for determining the enzymatic activity of pectinase.

2. Scope

This procedure applies to Product Numbers P4716, P0690, P2401, and P4300.

3. Definitions

3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC

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 25 ºC.

4. Discussion

Polygalacturonic Acid + H₂O    ^Pectinase   > Galacturonic Acid
Polygalacturonic Acid + I₂      > Oxidation products
2Na₂S₂O₃+ I₂      > 2NaI + Na₂S₄O₆
The excess Iodine is titrated with Sodium Thiosulfate.

5. Responsibilities

Analytical services laboratory personnel should follow this procedure as written.

6. Safety

Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:

T = 25 °C, pH = 4.0

7.2 METHOD:
Titrimetric Stop Reaction

7.3 REAGENTS:

7.3.1 
0.5%(w/v) Polygalacturonic Acid (Sub)

7.3.1.1
Prepare by adding 200 mL of purified water to 1.00 g to 1.05 g of polygalcturonic acid (Product No. P3889) with stirring.

7.3.1.2
With stirring, bring to a boil and maintain boiling for exactly five minutes. Immediately place in a 25 ºC water bath with an immersible stirrer. Stir until the temperature is at 25 ºC and allow the solution to stir for an additional ten minutes.

7.3.1.3
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.

7.3.1.4
Recheck pH and adjust with Reagent 7.3.3 (NaOH) at 25 ºC 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.

7.3.1.5
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 25 ºC. If not, adjust pH with 0.1N NaOH.

7.3.2
100 mM Iodine (I₂)
Use Iodine volumetric Standard, 0.1 N (Product No. 318981).

7.3.3
1.0 N Sodium Hydroxide (NaOH) Solution (Product No. S2567).

7.3.4
1 M Sodium Carbonate (Na₂CO₃)
Prepare in purifed water at 106 mg/mL using Sodium Carbonate, Reagent Grade (Product No. S2127).

7.3.5
2.0 N Sulfuric Acid (H₂SO₄)
Prepare in purified water at 0.06 mL/mL using Sulfuric Acid, ACS Reagent (Product No. 258105).

7.3.6
10 N Sodium Hydroxide (NaOH-2)
Prepare at 400 mg/mL in purified water using Sodium Hydroxide, Reagent Grade (Product No. S5881).

7.3.7
100 mM Sodium Thiosulfate, Standardized (Na₂S₂O₃)
Prepare in 3 L of purifed water using 78.3 g of Sodium Thiosulfate, Pentahydrate (Product No. S8503) and 0.6 g of Sodium Carbonate, Monohydrate (Product No. 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 mg/mL by boiling for exactly five minutes with stirring. Cool to room temperature. Use starch, potato soluble (Product No. 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:

	Test	Blank
Reagent 7.3.1 (Sub)	4.90	4.90

7.4.2    Mix by swirling and equilibrate to 25 ºC 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 25 ºC. 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 CALCULATIONS

7.5.1	Units/mL enzyme  =	(mLs of titrant for blank - mLs of titrant for test)(1)(df)(100)
		(0.100)(5.0)(2)

where:
    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, the final concentrations are 0.49%(w/v) Polygalacturonic Acid and 10 units of Pectinase.

8. References

1. Kertesz Z. 1955. Methods in Enzymology. 1162-164.