General description
β-Glucuronidase/Arylsulfatase from Helix pomatiais is the most widely used enzyme preparation containing sulfatase activity and has a very broad specificity. It is a crude mixture of enzymes and the preparation does not involve any kind of chromatographic separation. This preparation contains both glucuronidase and sulfatase activity. Enzymatic hydrolysis prior to detection is essential for achieving high sensitivity during analysis. After hydrolysis, the sample may be analyzed by mass spectroscopy, gas chromatography, HPLC, or immunoassays. For doping analysis, the sulfatase activity in addition to the glucuronidase activity is essential for the detection of all drug conjugates present in the sample. β-Glucuronidase/Arylsulfatase is also used for the enzymatic hydrolysis of glucuronides and sulfate esters in biological fluids and primarily urine.
Application
β-Glucuronidase/Arylsulfatase has been used extensively in research and analytic laboratories for the simultaneous enzymatic hydrolysis of steroid β-glucuronides and sulfate esters. The enzyme is used during sample preparation to cleave off glucuronides and sulfate esters prior to GC-MS, HPLC, immunoassays, or other analytical methods, including doping analysis and hydrolysis of steroid conjugates (glucuronides) and sulfate esters in urine and other body fluids.
Biochem/physiol Actions
β-Glucuronidase/Arylsulfatase shows a broad specificity for many kinds of β-glucuronides and sulfate esters.
Specificity of β-Glucuronidase:
The glycosides that β-D-glucuronic acid forms with a variety of compounds containing hydroxyl groups hydrolyze readily in the presence of β-glucuronidase.
Such compounds include steroids, such as estriol (Km = 0.42 mM, pH 4.5), androsterone, pregnanediol, tetrahydrocortisone,
phenols, such as phenolphthalein (Km = 0.39 mM), 4-nitro-phenol, 4-methylumbelliferone,
drugs such as chloramphenicol and tetrahydrocannabinols,
and metabolites such as thyroxine and bilirubin.
Polysaccharides that contain β-glucuronic acid residues, such as hyaluronic acid, are also hydrolyzed.
β-Glucuronidase is highly specific for the carbohydrate part: neither α-glucosides nor β-glucosiduronic acids are hydrolyzed. However, the nature of the residue linked to the β-glucuronic acid residue is hardly important at all.
Specific activity: 5.5 U/ml at +38°C with phenolphthalein-β-glucuronide as the substrate (4.5 U/ml at +25°C with 4-nitro-phenyl-β-D-glucuronide as the substrate = 100,000 Fishman units/ml at +38°C with phenolphthalein-β-glucuronide as the substrate). 1 Fishman unit releases 1 μg phenolphthalein from phenolphthalein-β-glucuronide in 1 hour at +38°C.
Specificity of Acrylsulfatase:
Sulfate esters of many phenols are hydrolysed in the presence of arylsulfatase. Examples are steroid sulfates such as estronesulfate, 4-nitrophenyl hydrogen sulfate (Km = 1.8 mM, pH 7.3), 4-nitro-pyrocatechol 2-sulfate (Km = 1.25 mM, pH 7.5), and phenolphthalein disulfate.
Specific activity: 2.6 U/ml at +38°C with phenolphthalein disulfate as the substrate (14 U/ml at +25°C with 4-nitrophenyl sulfate as the substrate = 800,000 Roy units/ml at +38°C with 2-hydroxy-5-nitrophenyl sulfate as the substrate). 1 Roy unit releases 1 μg 2-hydroxy-5-nitrophenyl sulfate in 1 hour at +38°C.
Specificity of β-Glucuronidase:
The glycosides that β-D-glucuronic acid forms with a variety of compounds containing hydroxyl groups hydrolyze readily in the presence of β-glucuronidase.
Such compounds include steroids, such as estriol (Km = 0.42 mM, pH 4.5), androsterone, pregnanediol, tetrahydrocortisone,
phenols, such as phenolphthalein (Km = 0.39 mM), 4-nitro-phenol, 4-methylumbelliferone,
drugs such as chloramphenicol and tetrahydrocannabinols,
and metabolites such as thyroxine and bilirubin.
Polysaccharides that contain β-glucuronic acid residues, such as hyaluronic acid, are also hydrolyzed.
β-Glucuronidase is highly specific for the carbohydrate part: neither α-glucosides nor β-glucosiduronic acids are hydrolyzed. However, the nature of the residue linked to the β-glucuronic acid residue is hardly important at all.
Specificity of Acrylsulfatase:
Sulfate esters of many phenols are hydrolysed in the presence of arylsulfatase. Examples are steroid sulfates such as estronesulfate, 4-nitrophenyl hydrogen sulfate (Km = 1.8 mM, pH 7.3), 4-nitro-pyrocatechol 2-sulfate (Km = 1.25 mM, pH 7.5), and phenolphthalein disulfate.
Glucuronidation is one of the basic principles of metabolism. Most 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.
In addition, sulfation of the drug or chemical substance by sulfotransferases in the human body may occur. Sulfation in general, is less predictable than glucuronidation since many isoenzymes of sulfotransferases exist, and there are individual differences in the ratio of glucuronidation versus sulfation of the substance.
Features and Benefits
Deconjugate and detect both glucuronides and sulfate esters.
Quickly screen for steroids, benzodiazepines, cannabinoids, opioids, and other drugs.
Physical form
Solution in saline, stabilized
Preparation Note
Working concentration: In many applications the product can be diluted with water immediately before use or employed undiluted.
Note: The present β-glucuronidase/arylsulfatase preparation is very concentrated and must be diluted for some applications. Moreover, in the preparation of protoplasts, the precise concentration to use for a given strain of yeast must be found empirically.
Storage conditions (working solution): Note: Aliquot portions of the diluted preparation may be stored at -15 to -25 °C; they should not be thawed and refrozen more than a time or two, and storage at the lower temperature does not lengthen their life beyond that of the product kept at 2 to 8 °C.
Avoid repeated freezing and thawing (more than 2 times).
Other Notes
Glucuronidase:
Standard unit
The standard unit of β-glucuronidase activity is the enzyme activity that increases the rate of release of 4-nitrophenol from 4-nitrophenyl β-D-glucosiduronic acid at a temperature of +25 °C and pH 4.5 by 1 μM.
Phenolphthalein unit
The phenolphthalein unit of β-glucuronidase activity is the enzyme activity that increases the rate of release of phenolphthalein from phenolphthalein β-D-glucosiduronic acid at a temperature of +38 °C by 1 μM.
Approx. 4.5 standard units are equivalent to 5.5 phenolphthalein units.
Fishman unit
The Fishman unit of β-glucuronidase activity is the enzyme activity that increases the rate of release of phenolphthalein from phenolphthalein β-D-glucosiduronic acid at a temperature of +38 °C by 1 μg.
Approx. 1 standard unit is equivalent to 22,000 Fishman units (1 phenolphthalein unit is equivalent to 19,000 Fishman units).
Arylsulfatase:
Standard unit
The standard unit of arylsulfatase activity is the enzyme activity that increases the rate of release of 4-nitrophenol from 4-nitrophenyl sulfate at a temperature of +25 °C and pH 6.2 by 1 μM.
Phenolphthalein unit
The phenolphthalein unit of arylsulfatase activity is the enzyme activity that increases the rate of release of phenolphthalein from phenolphthalein disulfate at a temperature of +38 °C and pH 6.2 by 1 μM.
Approx. 5.4 standard units are equivalent to 1 phenolphthalein unit.
Roy unit
The Roy unit of arylsulfatase activity is the enzyme activity that increases the rate of release of 4-nitropyrocatechol from 2-hydroxy-5-nitrophenyl hydrogen sulfate (4-nitropyrocatechol 2-sulfate) at a temperature of +38 °C and pH 6.2 by 1 μg.
Approx. 1 standard unit is equivalent to 57,000 Roy units (1 phenolphthalein unit is equivalent to 308,000 Roy units).
EC 3.1.6.1 & 3.2.1.31
For life science research only. Not for use in diagnostic procedures.
