Glucuronidase, Beta Assay

Method: As described by Fishman et al. (1948) utilizing phenolphthalein glucuronidate as substrate. One unit hydrolyzes one micromole of phenolphthalein glucuronidate per minute at 37°C and pH 4.5 under the specified conditions. One Worthington unit is equivalent to 19,000 Fishman units.

Reagents

• 0.15 M Sodium chloride. Keep on ice.
• 0.1 M Sodium acetate, pH 4.5
• 0.01 M Phenolphthalein glucuronidate, pH 7.0
• 0.2 M Glycine with 0.2 M Sodium chloride, pH 10.4

Enzyme

Dissolve enzyme at a concentration of 2 mg/ml in cold 0.15 M sodium chloride. For assay dilute enzyme to a concentration ranging from 0.25 to 2 mg/ml.

Procedure

Into a series of numbered tubes pipette as follows: (include one tube as a blank).

0.1 M Sodium acetate	0.8 ml
0.01 M Phenolphthalein glucuronidate	0.1 ml

Incubate in 37°C water bath for five minutes to attain temperature equilibration. At timed intervals add to each tube, 0.1 ml of an enzyme dilution. To the blank, add 0.1 ml water.

Incubate for 30 minutes. Stop the reaction by adding 5.0 ml of 0.2 M glycine, 0.2 M NaCl, pH 10.4.

Remove from the water bath, cool to room temperature and read A₅₄₀ vs. the blank.

Calculation

Determine micromole phenolphthalein released from a standard curve.