Sigma β-Glucuronidases are routinely used for the enzymatic hydrolysis of glucuronides from urine,1,2 plasma,3,4 and other fluids5 prior to analysis by enzyme immunoassay, mass spectrometry, gas chromatography, high performance liquid chromatography, or other means. Typically, between 1 and 20 units of glucuronidase is used per µl of plasma, urine, or bile for the enzymatic hydrolysis of glucuronides present in these samples.1-5 The exact amount needed will depend on the specific conditions used and must be determined empirically.
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Glucuronidation, conjugation with glucuronic acid, by the human UDP-glucuronosyltransferase (UGT) family of enzymes plays an important role in the metabolic fate of many drugs and other xenobiotics. This biosynthetic reaction also has a role in the conjugation and excretion of endogenous substrates, such as steroids, bilirubin, and bile acids.6 UGT activity results in the conjugation of glucuronic acid to substrates containing sulfhydryl, hydroxyl, aromatic amino, or carboxylic acid moieties. The glucuronides formed are more polar (water soluble) than the parent organic substrate and are generally excreted through the kidney.
β-Glucuronidase (EC 3.2.1.31) catalyzes the reaction:
β-D-glucuronoside + H2O <--> D-glucuronate + an alcohol
One Sigma or modified "Fishman" unit will liberate 1.0 µg of phenolphthalein from phenolphthalein glucuronide per hr at 37°C at pH 5.0 (pH 6.8 for the E. coli source) (30 min assay).
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Molluskan Source β-Glucuronidases
β-Glucuronidase preparations isolated from mollusks also contain sulfatase activity. For this reason, the sulfatase activity of these preparations is also reported.
One unit of sulfatase will hydrolyze 1.0 mmole p-nitrocatechol sulfate per hr at pH 5.0 at 37 °C.
| |
Optimal pH |
|
| |
glucuronidase activity: |
4.5 to 5.0 |
| |
sulfatase activity: |
~6.2 |
| Molluskan Source Products |
| Product Type |
Form |
Activity |
Product # |
| Type HA-4 from Helix aspersa |
powder |
250,000-500,000 units/g solid,
up to 7,500 units/g solid sulfatase |
G4259 |
| Type HP-2 from Helix pomatia |
solution |
minimum 100,000 units/mL,
up to 7,500 units/mL sulfatase |
G7017 |
| Type H-2 from Helix pomatia |
solution |
minimum 85,000 units/mL,
up to 7,500 units/mL sulfatase |
G0876 |
| Type HP-2S from Helix pomatia |
sterile-filtered solution |
1,000-5,000 units/mL,
1,000-5,000 units/mL sulfatase |
G7770 |
| Type H-1 from Helix pomatia |
lyophilized powder |
minimum 300,000 units/g solid,
minimum 100,000 units/g solid sulfatase |
G0751 |
| Type H-3 from Helix pomatia |
solution |
~ 100,000 units/mL,
up to 1,000 units/mL sulfatase |
G8885 |
| Type H-5 from Helix pomatia |
lyophilized powder |
minimum 400,000 units/g solid
up to 40,000 units/g solid sulfatase
(A further purification of G0876) |
G1512 |
| Type H-3AF from Helix pomatia |
solution |
minimum 60,000 units/mL
(A further purification of Type H-3 to remove agglutinin) |
G0762 |
| Type H-5 from Patella vulgata |
lyophilized powder |
1,000,000-3,000,000 units/g solid |
G8132 |
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Bovine Liver Source β-Glucuronidases
Bovine β-Glucuronidase is a 290 kD protein with an isoelectric point of 5.1.7
Bovine preparations typically contain small amounts of sulfatase activity, usually less than 0.5%.
| |
Optimal pH |
|
| |
glucuronidase activity: |
4.4 |
| |
sulfatase activity: |
4.4 |
| Bovine Liver Source Products |
| Product Type |
Form |
Activity |
Product # |
| Type B-1 |
lyophilized powder |
minimum 1,000 units/mg solid,
~85% protein |
G0251 |
| Type B-3 |
lyophilized powder |
~3,000 units/mg solid,
~50% protein |
G0376 |
| Type B-10 |
lyophilized powder |
~10,000 units/mg solid |
G0501 |
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E. coli Source β-Glucuronidases
β-Glucuronidase is a ~290 kDa tetrameric protein with an isoelectric point of 4.8.8 Unlike the enzyme preparations from mollusks that naturally contain β-glucuronidase and sulfatase activities in almost equal amounts, the preparation of β-glucuronidase from E. coli is essentially free of sulfatase activity. The enzyme from E. coli has a high rate of hydrolytic activity and it retains this activity during hydrolysis better than similar enzymes that are more sensitive to changes in the concentration of β-glucuronide conjugates. The enzyme preparation from E. coli has been shown to be useful for determining the presence of androsterone, 17-hydroxycorticosteroids, and estriol in urine.9 The E. coli enzyme has also been shown to be more active against estrogen conjugates than other sources of the enzyme.10
| E. coli Source Products |
| Product Type |
Form |
Activity |
Product # |
| New! Recombinant, from overexpressing E. coli |
lyophilized powder |
minimum 10,000,000 units/g protein |
G8295 |
| New! Recombinant, from overexpressing E. coli |
lyophilized powder |
minimum 20,000 units/mg protein |
G8420 |
| Type IX-A |
lyophilized powder |
1,000,000-5,000,000 units/g protein,
~50% protein |
G7396 |
| Type VII-A |
lyophilized powder |
5,000,000-20,000,000 units/g protein,
~25% protein |
G7646 |
| Type X-A |
lyophilized powder |
20,000,000-60,000,000 units/g protein,
~30% protein |
G7896 |
| Aqueous Glycerol Solution |
aqueous glycerol solution |
20,000,000-60,000,000 units/g protein |
G8162 |
| Preweighed Vials |
lyophilized powder,
(min. 1,000 units per vial) |
5,000,000-20,000,000 units/g protein,
when reconstituted with 10 mL water, solution will contain ~4 mM phosphate buffer, pH 6.8. |
G8396 |
| Preweighed Vials |
lyophilized powder,
(min. 1,000 units per vial) |
5,000,000-20,000,000 units/g protein |
G8271 |
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Related Products
| Inhibitors |
| Product Name |
Product # |
| D-Glucuronic acid |
G5269 |
| D-Glucaro-δ-lactam potassium salt |
G7166 |
| ψ-Tectorigenin |
T9165 |
| Substrates |
| Product Name |
Product # |
| 5-Bromo-6-chloro-3-indolyl β-D-glucuronide |
B4532 |
| 5-Bromo-4-chloro-3-indolyl β-D-glucuronide |
B8174 |
| 8-Hydroxyquinoline glucuronide |
H1254 |
| 4-Methylumbelliferyl β-D-glucuronide |
M5664 |
| 4-Nitrophenyl β-D-glucopyranoside |
N1627 |
| Phenolphthalein β-D-glucuronide |
P0501 |
| Phenolphthalein β-D-glucuronide sodium salt |
P0376 |
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References
- Xu, X., et al., J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 780, 315-330 (2002).
- Staimer, N., et al., Anal. Chim. Acta, 444, 27-36 (2001).
- Zhai, P., et al., Am. J. Physiol. Heart Circ. Physiol., 281, H1223-H1232 (2001).
- Anderson, A. et al., Cancer Chemother. Pharmacol., 44, 422-426 (1999).
- Nobilis, M., J. Chromatogr. A., 1031, 229-236 (2004).
- Tephly, T.R., et al., Adv. Pharmacol., 42, 343-346 (1998).
- Himeno, et al., J. Biochem.(Tokyo), 76, 1243 (1974)
- Kim, D-H, et al., Biol. Pharm. Bull., 18, 1184-1188 (1995).
- Graef, V., et al., Clin. Chem., 23, 532-535 (1977).
- Jayle, M.F., Scholler, R., Jarrige, P. & Mtay, S. Bull. Soc. Chim. Biol. 41, 1593. (1959)
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