To standardize a procedure for the enzymatic assay of β-Glucuronidase.
The scope of this procedure is all products from E. coli that have a specification for β-Glucuronidase activity.
One modified "Fishman" unit will liberate 1.0 μg of phenolphthalein from phenolphthalein glucuronide per hour at pH 6.8 at 37 °C.
Phep-Gluc + H2O β-Glucuronidase > D-Glucuronate + Phenolphthalein
PheP-Gluc = Phenolphthalein Glucuronide
Analytical services personnel should follow this protocol as written.
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.
T = 37 °C, pH = 6.8, A540nm, Light path = 1 cm
Spectrophotometric Stop Rate Determination
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.
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.
Corrected ΔA540 Standard = A540 Standard - A540 Standard blank
Prepare a standard curve by plotting the ΔA540 for the Standard vs micrograms of Phenolphthalein.
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|
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.