ß-Glucuronidase Enzymatic Assay

1. Objective

To standardize a procedure for Enzymatic Assay of b-Glucuronidase¹.

2. Scope

This procedure applies to all b-Glucuronidase products that are derived from Helix Pomatia and Bovine Liver.

3. Definitions

One Sigma or modified "Fishman" unit will liberate 1.0 microgram of Phenolphthalein from Phenolphthalein Glucuronide per hour at pH 5.0 at 37 °C.

4. Discussion

PheP-Gluc + H₂O   ^{b-Glucuronidase}   >D-Glucuronate + Phenolphthalein

Abbreviation:
PheP-Gluc = Phenolphthalein Glucuronide

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. Safety

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

7. Procedure

7.1 CONDITIONS:
T = 30 °C, pH = 5.0, A_540nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Stop Rate Determination

7.3 REAGENTS:

7.3.1    100 mM Sodium Acetate Buffer, pH 5.0 at 37 °C
(Prepare 50 mL in deionized water using Sodium Acetate, Trihydrate. Adjust to pH 5.0 at 37°C with 1 M HCl.)

7.3.2    1.2 mM Phenolphthalein Glucuronide Substrate Solution (PheP-Gluc)
(Prepare by diluting 1 mL of Phenolphthalein Glucuronic Acid, Stock Number 105-4, with 8 mL of deionized water.)

7.3.3    200 mM Glycine Buffer Solution, pH 10.4
(Prepare 100 mL in deionized water using Glycine Free Base, Product Number G7126. Adjust to pH 10.4 at 37°C with 1 M NaOH.)

7.3.4    0.2% (w/v) Sodium Chloride Solution (NaCl)
(Prepare 20 mL in deionized water using Sodium Chloride.)

7.3.5    b-Glucuronidase Enzyme Solution
(Immediately before use, prepare a solution containing 250-500 units/mL of b-Glucuronidase in cold Reagent 7.3.4)

7.3.6    95% (v/v) Ethanol
(Prepare 20 mL in deionized water using 200 Proof USP Ethyl Alcohol.)

7.3.7    0.05% (w/v) Phenolphthalein Standard Solution (Std Soln)
(Prepare 2 mL by dissolving 1.0 mg of Phenolphthalein in Reagent 7.3.6)

7.4 ASSAY:

7.4.1    Pipette (in milliliters) the following reagents into suitable containers:

	Test	Blank
Reagent A (Buffer)	0.70	0.70
Reagent B (PheP-Gluc)	0.70	0.70

7.4.2    Mix by inversion and equilibrate to 37 °C. Then add:

	Test	Blank
Reagent E (Enzyme Solution)	0.10	-----

7.4.3    Mix by inversion and incubate at 37 °C for exactly 30 minutes. Then add:

	Test	Blank
Reagent C (Glycine Buffer)	5.00	5.00
Reagent E (Enzyme Solution)	-----	0.10

7.4.4    Immediately mix by inversion. Transfer the solutions to suitable cuvettes and record the A_540nm for both the Test and Blank using a suitable spectrophotometer.

7.4.5    STANDARD CURVE:

Prepare a standard curve by pipetting (in milliliters) the following reagents into suitable containers:

	Std 1	Std 2	Std 3	Std 4	Blank
Reagent A (Buffer)	0.70	0.70	0.70	0.70	0.70
Reagent B (PheP-Gluc)	0.70	0.70	0.70	0.70	0.70
Reagent G (Std Soln)	0.03	0.05	0.07	0.10	-----
Reagent F (Ethanol)	0.07	0.05	0.03	-----	0.10
Reagent C (Glycine Buffer)	5.00	5.00	5.00	5.00	5.00

7.4.6    Mix by inversion and transfer the standards to suitable cuvettes. Record the A_540nm for each standard using a suitable spectrophotometer.

8. Calculations

8.1    Standard Curve:

ΔA₅₄₀ Standard = A₅₄₀ Standard - A₅₄₀ Standard blank
Prepare a standard curve by plotting the ΔA₅₄₀ for the Standard vs micrograms of Phenolphthalein.

8.2    Sample Determination:

ΔA₅₄₀ Sample = A₅₄₀ Sample - A₅₄₀ Sample blank
Determine the total micrograms of phenolphthalein liberated using the Standard curve.

Units/mL enzyme =	(mg phenolphthalein released)(2)(df)
	0.1

 

2 = Conversion factor from 30 minutes to 1 hour as per the Unit Definition
df = Dilution factor
0.1 = Volume (in milliliter) of enzyme used

Units/mg solid =	Units/mL enzyme
	mg solid/mL enzyme

 

Units/mg protein =	Units/mL enzyme
	mg protein/mL enzyme

9. Final Assay Contentration

In a 1.50 mL reaction mix, the final concentrations are 47 mM Sodium Acetate, 0.56 mM Phenolphthalein Glucuronic Acid, and 25-50 units b-Glucuronidase.

11. Notes

11.1    This assay is not to be used to assay b-Glucuronidase, Insoluble Enzyme attached to beaded Agarose.

11.2    This assay is based on the cited references.

11.3    History indicates product is fairly stable.

11.4    Use P9750 as Phenolphthalein Standard, or Standard Solution 105-1 while supplies last.

11.5    We warrant that the above procedure information is currently utilized by our quality control teams and that our products conform to the information in this publication. Purchaser must determine the suitability of the information and products for its particular use. Upon purchase, see reverse side of invoice or packing slip for additional terms and conditions of sale.

12. Approval

	Print Name	Sign Name	Title	Date
Prepared by:	Carrie Cupp	Carrie Cupp	Chemist	7/20/04
Approved by:	David Lintz	David Lintz	Supervisor, Analytical Services	8/13/04
Approved by:	Gene McNaughton	Gene McNaughton	Quality Assurance	8/16/04