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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 Sigma or 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, A540nm, 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, Sigma Prod. No. P-5379 and Albumin, Bovine, Sigma 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, Sigma Prod. No. P-0501.
  • 200 mM Glycine Buffer Solution, pH 10.4.
    Use Glycine Buffer Solution, Sigma Stock No. 105-2, or prepare 100 mL in deionized water using Glycine Free Base, Sigma 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, Sigma Prod. No. P-9750 in 5 mL of Reagent E or use Phenolphthalein Standard Solution, Sigma 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 Sigma Product or Stock numbers are specified, equivalent reagents may be substituted.

 

8. References & Attachments

Fishman, W.H. and Bernfeld, P. (1955) Methods in Enzymology, Volume I, 262-269
Combie, J., Blake, J.W., Nugent, T.E., and Tobin, T. (1982) Clin. Chem. 28, 83-86
Fishman, W.H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U. ed) 2nd ed., Volume II, 930-932, Academic Press, New York, NY

9. 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

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