Enzymatic Assay of β-Glucuronidase (EC 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

Analytical services personnel should follow this protocol as written.

6. Safety

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

7. Procedure


T = 37 °C, pH = 6.8, A540nm, Light path = 1 cm


Spectrophotometric Stop Rate Determination


  • 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, Product No. P5379 and albumin, bovine, Product No. A4503. Adjust to pH 6.8 at 37 °C with 1 M KOH.
  • 3.0 mM Phenolphthalein Glucuronide Substrate Solution (PheP-Gluc)
    Prepare 10 mL in deionized water using Phenolphthalein Glucuronic Acid, Free Acid, Product No. P0501.
  • 200 mM Glycine Buffer Solution, pH 10.4.
    Use Glycine Buffer Solution, Product No. 105-2, or prepare 100 mL in deionized water using Glycine Free Base, Product No. G7126. 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, Product No. P9750 in 5 mL of Reagent E or use Phenolphthalein Standard Solution, Product No. 105-1.

Test Method:

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

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

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

Immediately mix by inversion. Transfer the solutions to suitable cuvettes and record the absorbance at 540 nm 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:

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


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)

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.


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

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