Enzymatic Assay of β-Glucuronidase (EC 3.2.1.31) from E. coli

1. Objective

To standardize a procedure for the enzymatic assay of β-Glucuronidase.

2. Scope

The scope of this procedure is all products from E. coli that have a specification for β-Glucuronidase activity.

3. Definitions

One modified "Fishman" unit will liberate 1.0 μg of phenolphthalein from phenolphthalein glucuronide per hour at pH 6.8 at 37 °C.

4. Discussion

Phep-Gluc + H₂O ^{β-Glucuronidase} > D-Glucuronate + Phenolphthalein
Abbreviation used:
PheP-Gluc = Phenolphthalein Glucuronide

5. Responsibilities

It is the responsibility of all Analytical Services and QA personnel to follow this protocol as written.

6. Safety

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

7. Procedure

Conditions:

T = 37 °C, pH = 6.8, A_540nm, Light path = 1 cm

Method:

Spectrophotometric Stop Rate Determination

Reagents:

• 75 mM Potassium Phosphate Buffer, with 1.0% (w/v) Bovine Serum Albumin, pH 6.8 at 37 °C
  Prepare 100 mL in deionized water using Potassium Phosphate, Monobasic, Anhydrous, Prod. No. P-5379 and Albumin, Bovine, Prod. No. A-4503. Adjust to pH 6.8 at 37 °C with 1M KOH.
• 3.0 mM Phenolphthalein Glucuronide Substrate Solution (PheP-Gluc)
  Prepare 10 mL in deionized water using Phenolphthalein Glucuronic Acid, Free Acid, Prod. No. P-0501.
• 200 mM Glycine Buffer Solution, pH 10.4.
  Use Glycine Buffer Solution, Stock No. 105-2, or prepare 100 mL in deionized water using Glycine Free Base, Prod. No. G-7126. Adjust to pH 10.4 at 37 °C with 1 M NaOH.
• β-Glucuronidase Enzyme Solution
  Immediately before use, first prepare a 5 mg/mL solution of β-Glucuronidase in cold Reagent A. Then dilute to a final concentration of 400 - 800 units/mL in cold reagent A.
• 95% (v/v) Ethanol
  Prepare 20 mL in deionized water using 200 Proof USP Ethyl Alcohol, Quantum Chemical Corporation.
• 0.05% (w/v) Phenolphthalein Standard Solution (Std Soln)
  Prepare 5 mL by dissolving 2.5 mg of Phenolphthalein, Prod. No. P-9750 in 5 mL of Reagent E or use Phenolphthalein Standard Solution, Stock No. 105-1.

Test Method:

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

	Test	Blank
Deionized Water	0.65	0.65
Reagent A (Buffer)	0.50	0.50
Reagent B (PheP-Gluc)	0.25	0.25

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

Reagent D (Enzyme Solution)

0.10

------

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

Reagent C (Glycine Buffer)	5.00	5.00
Reagent D (Enzyme Solution)	------	0.10

Immediately mix by inversion. Transfer the solutions to suitable cuvettes and record the A540nm for both the Test and Blank using a suitable spectrophotometer.

Standard Curve:

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

	Std 1	Std 2	Std 3	Std 4	Std 5	Blank
Deionized Water	0.65	0.65	0.65	0.65	0.65	0.65
Reagent A (Buffer)	0.50	0.50	0.50	0.50	0.50	0.50
Reagent B (PheP-Gluc)	0.25	0.25	0.25	0.25	0.25	0.25
Reagent F (Std Soln)	0.02	0.03	0.05	0.07	0.10	----
Reagent E (Ethanol)	0.08	0.07	0.05	0.03	----	0.10
Reagent C (Glycine Buffer)	5.00	5.00	5.00	5.00	5.00	5.00

Mix by inversion and transfer the standards to suitable cuvettes. Record the A540nm for each standard using a suitable spectrophotometer.

Calculations:

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

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

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

2 = Time correction of assay (Unit Definition = 1 hour)
df = Dilution factor
0.1 = Volume (in milliliters) of enzyme used

Units/g solid =	units/mL enzyme
	g solid/mL enzyme

Units/g protein =	units/mL enzyme
	g protein/mL enzyme

Final Assay Concentration:

In a 1.50 mL reaction mix, the final concentrations are 30 mM potassium phosphate, 0.50 mM phenolphthalein glucuronic acid, and 40 - 80 units β-glucuronidase.

NOTES:

1. This assay is based on the cited references.
2. Where our Product or Stock numbers are specified, equivalent reagents may be substituted.

8. Approval

Print Name	Sign Name	Title	Date
Prepared by: Kristen VonGruben	Kristen VonGruben	Originator	09-18-02
Approved by: David Lintz	David Lintz	Supervisor, Analytical Services	09-06-02
Approved by: Jeff Heiland	Jeff Heiland	Quality Assurance	09-24-02