Glucuronidation by the human UDP-glucuronosyltransferase (UGT) family of enzymes is important 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. 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. Similar conjugation reactions occur with isoforms of sulfotransferases yielding the sulfate conjugate.
β-Glucuronidases are routinely used for the enzymatic hydrolysis of glucuronides from urine, plasma, and other biological fluids before analysis by enzyme immunoassay, mass spectrometry, gas chromatography, high performance liquid chromatography, or other means (Figure 1). 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. The exact amount needed will depend on the specific conditions used and must be determined empirically.
Figure 1. β-Glucuronidase is routinely used to hydrolyze xenobiotic glucuronide metabolites to increase volitility and organic solvent solubility prior to GC analysis.
ß-Glucuronidase preparations isolated from mollusks also contain sulfatase activity. For this reason, the sulfatase activity of these preparations is also reported on the certificate of analysis.
The enzyme from patella vulgata is reported to be much more effective in hydrolyzing opioidglucuronides. Whereas the Helix pomatia and E. coli enzymes are reported to be slightly more effective in hydrolyzing steroidglucuronides.
E. coli derived β-glucuronidase is a ~290 kDa tetrameric protein with an isoelectric point of 4.8. 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. The E. coli enzyme has also been shown to be more active against estrogen conjugates than other sources of the enzyme.
Optimal pH: 6–7
Bovine ß-glucuronidase is a 290 kD protein with an isoelectric point of 5.1.
Bovine preparations typically contain small amounts of sulfatase activity, usually less than 0.5%.
β-glucuronidase activity: 4.4
sulfatase activity: 4.4
Molluskan sources of sulfatase also contain β-glucuronidase activity. Lot-specific activities are reported for both enzymes on the certificate of analysis.
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