b-Glucuronidase has been found in tissue extracts of mammals and other vertebrates, digestive juices of snails, molluscs, locusts, bacteria and plants. The enzyme catalyzes the hydrolysis of b-glucuronides and also the transfer of glucuronly radicals to acceptor alcohols. b-Glucuronidase is a structural protein of the endoplasmic reticulum and its occurance in the lysozomes is due to changes in the endoplasmic reticulum.
b-Glucuronidase catalyzes the following reaction:b-Glucuronidase b-D-Glucuronide + H2O ---> Alcohol + D-Glucuronate
Solubility:Readily soluble in ddH20 or dilute buffer.
Activity: ~~0.1-5u/mg protein
Protein: 10mg/ml
Unit Definition:1 unit is equal to the amount of enzyme which catalyzes the formation of one micromole of p-Nitrophenol per minute at 37°C.
Quality Control:SDS-PAGE
Optimum pH:4.4
Extinction Coefficient: 17.0
Isoelectric Point:5.1
Specificity:The enzyme hydrolyzes a large variety of conjugated glucuronides but not alpha-or beta-glucosides.
Inhibitors:Strongly, reversibly inhibited by various organic peroxides
Storage and Stability:Lyophilized powder may be stored at -20°C. Stable for 12 months at -20°C. Reconstitute with sterile buffer or ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Assay:0.1M Acetate buffer, pH 4.50.1M p-Nitrophenyl glucuronideEnzyme solution in distilled water0.5 N NaOH.
1.Set up spectrophotometer at 405 nm and 37°C. 2.Place the following reagents in a test tube and equilibrate in the water bath at 37°C for about 15 minutes:0.1M Acetate buffer, pH 4.5, 1ml p-Nitrophenyl glucuronide, 0.03ml
3.Add 0.03ml of the enzyme solution and mix
4.After the test tubes have incubated for exactly 10 minutes at 37°C, add 2ml of 0.5N NaOH solution to stop the reaction (ODtest). At the same time, prepare the blank by first mixing the reagent mixture with 2ml of NaOH solution after the 10 minute incubation period at 37°C, and then adding the enzyme solution (ODblank)
5.Measure the absorbance of the test (ODtest) and blank (ODblank) at 405nm against H2O.
