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β-Glucuronidase

from E. coli K 12

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Properties

Related Categories β-Glucuronidase (beta-Glucuronidase), E. coli Source, Analytical and Industrial Enzymes, Biochemicals and Reagents, Enzymes, Inhibitors, and Substrates More...
Quality Level   100
form   solution
specific activity   ~80 units/mg protein (Glucuronidase at 25 °C, or 140 U/mg at 37 °C, at pH 7 with 4-nitrophenyl-β-D-glucuronide as substrate; 1 ml β-Glucuronidase contains at least 140 U at 37 °C.)
mol wt   Mr ~220 kDa
packaging   pkg of 1 mL (03707580001)
  pkg of 15 mL (03707601001)
  pkg of 5 mL (03707598001)
mfr. no.   Roche
parameter   48 °C optimum reaction temp.
optimum pH   6.0-6.5
shipped in   wet ice
storage temp.   2-8°C

Description

General description

β-D-Glucuronoside glucuronosohydrolase

Glucuronidation is one of the basic principles of metabolism. Many substances presented to the human body undergo metabolic processing that includes conjugation with glucuronic acid by UDP-glucuronosyltransferases (UGTs). This enzyme catalyzes the transfer of a glucuronyl group to many biological and pharmacologically active endogenous and exogenous molecules. Glucuronide is in general, more soluble, less toxic, and more easily excreted by the human body compared to the original molecule. To analyze these drug conjugates that are present in body fluids, such as urine and plasma, deconjugation of the glucuronide is necessary.
Substances used for doping are conjugated with glucuronides, which means that effective doping analysis relies very often on the enzymatic cleavage of conjugated drug molecules by β-glucuronidase.

Enzyme Characteristics
The E. coli β-glucuronidase enzyme is essentially free of sulfatase activity and is especially efficient in cleaving steroid and benzodiazepine conjugates.
β-D-Glucuronoside glucuronosohydrolase
EC 3.2.1.31

Specificity

Cleaves terminal glucuronic acid which is β-linked to mono-, oligo- or polysaccharides or phenol.

Application

β-Glucuronidases have been used extensively in research and analytical laboratories for the enzymatic hydrolysis of steroid β-glucuronides. β-Glucuronidase is used:
• for the hydrolysis of steroid conjugates (glucuronides) in urine (pH 6.0–6.5)
• in doping analysis
• for the detection of benzodiazepine in small doses.
• during sample preparation to cleave off glucuronides prior to GC-MS, HPLC, immunoassays, or other analytical methods.
It has been used for the hydrolysis of plasma enterolactone, testosterone and epitestosterone glucuronide.

Biochem/physiol Actions

Glucuronidation is one of the basic principles of metabolism. Many substances presented to the human body undergo metabolic processing that includes conjugation with glucuronic acid by UDP-glucuronosyltransferases (UGTs). β-Glucuronidase, also known as β-D-glucuronoside glucuronosohydrolase, catalyzes the transfer of a glucuronyl group to many biological and pharmacologically active endogenous and exogenous molecules. Glucuronide is in general, more soluble, less toxic, and more easily excreted by the human body compared to the original molecule. To analyze these drug conjugates that are present in body fluids, such as urine and plasma, deconjugation of the glucuronide is necessary. Enzymatic hydrolysis prior to detection is essential to achieving high sensitivity during analysis. After hydrolysis, the sample may be analyzed by mass spectroscopy, gas chromatography, HPLC, immunoassays, or other analytical methods.

Features and Benefits

β-Glucuronidase from E. coli is highly specific for β-glucuronides, and is capable of very quickly hydrolyzing steroid β-glucuronides as well as cleaving many other types of glucuronides, such as benzodiazepine, opioid, and cannabinoid. β-Glucuronidase is used for the enzymatic hydrolysis of glucuronides in biological fluids, primarily urine. Substances used for doping are conjugated with glucuronides, which means that effective doping analysis relies very often on the enzymatic cleavage of conjugated drug molecules by β-glucuronidase.

• Perform fast analysis due to the enzyme′s high specific activity.
• Quickly screen for steroids, benzodiazepines, cannabinoids, and opioids.
• Save time by developing your procedure without the need to clean up the reaction or buffer the urine.

Unit Definition

The international unit of β-glucuronidase activity is the enzyme activity that increases the rate of release of 4-nitrophenol from 4-nitrophenyl-β-D-glucuronide (4NPG) at a temperature of +25 °C and pH 7.0 by 1 μM.
The Fishman unit was previously used. This unit is the release of phenolphthalein from its glucuronide (PPG). It is not possible to measure the relative activities of different preparations for steroid β-glucuronides by comparing their activities with respect to PPG. Many preparation do not catalyze the hydrolysis of PPG, 4NPG, or the various steroid β-glucuronides in urine equally. The choice of 4NPG as standard substrate is based on the following considerations:
• The Michaelis concentrations for the two substrates are similar (KM = 2 ×10-4 M for 4 NPG and KM = 6 ×10-5 M for PPG), but the corresponding rates of hydrolysis differ:
• 4NPG is hydrolysed about 5 × as fast as PPG;
• For PPG, inhibition by excess substrate occurs; this is not observed using 4NPG.

Physical form

Solution in 50% glycerol, pH approximately 6.5
(15 ml in one bottle)

Other Notes

The E.coli β-glucuronidase enzyme is essentially free of sulfatase activity and is especially efficient in cleaving steroid and benzodiazepine conjugates.
For life science research only. Not for use in diagnostic procedures.

Legal Information

The sale of the Product does not exhaust or grant any rights in third party patents including patents of companies of the F. Hoffmann - La Roche AG group of companies, in particular, for the use of modified antibodies obtained by using the product.

Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport

Documents

Certificate of Analysis (COA)

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Protocols & Articles
Peer-Reviewed Papers
15

References

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