Inhibition of beta-glucuronidase (unknown origin) using p-nitrophenyl-beta-D-glucopyranosiduronic acid as substrate at 1 uM after 30 min by spectrophotometric analysis
Inhibition of beta-glucuronidase (unknown origin) using p-nitrophenyl-beta-D-glucuronide as substrate at 500 uM incubated for 30 min prior to substrate addition by spectrophotometric analysis
Inhibition of beta-glucuronidase (unknown origin) using p-nitrophenyl-beta-D-glucuronide as substrate incubated for 30 min prior to substrate addition by spectrophotometric analysis
Inhibition of beta-glucuronidase activity (unknown origin) assessed as p-nitrophenol formation after 30 mins using p-nitrophenyl-beta-D-glucuronide as substrate by spectrophotometry
Inhibition of beta-glucuronidase (unknown origin) at 500 uM using p-nitrophenyl-beta-D-glucuronide substrate incubated for 30 mins by spectrophotometry
Inhibition of beta-D-glucuronidase (unknown origin) assessed as reduction in p-nitrophenol formation using p-nitrophenyl-beta-D-glucuronide substarte incubated at 37 degC for 30 mins by spectrophotometric method
Inhibition of beta-glucuronidase (unknown origin) using p-nitrophenyl-beta-D-glucuronide as substrate assessed as formation of p-nitrophenol incubated for 30 mins by spectrophotometric analysis
Activity Assay: PNPG Assay: Two ß-glucuronidase activity assays were employed to examine the potency of inhibitors both in vitro and in cell-based studies. An absorbance assay based on the conversion of p-nitrophenyl glucuronide (PNPG) to p-nitrophenol (PNP) (Szasz (1967) Clin Chem 13:752-759), which absorbs at 410 nm, was employed as the primary in vitro assay. A secondary assay involving the conversion of phenolphthalein glucuronide (PheG) to phenolphthalein (Phe), which absorbs at 540 nm, was also employed. Id. Both assays were validated in vitro and in living cells, and the wavelengths monitored did not overlap with absorbance characteristics of putative inhibitors.
Cell-Based Assay: PheG Assay: Two ß-glucuronidase activity assays were employed to examine the potency of inhibitors both in vitro and in cell-based studies. An absorbance assay based on the conversion of p-nitrophenyl glucuronide (PNPG) to p-nitrophenol (PNP) (Szasz (1967) Clin Chem 13:752-759), which absorbs at 410 nm, was employed as the primary in vitro assay. A secondary assay involving the conversion of phenolphthalein glucuronide (PheG) to phenolphthalein (Phe), which absorbs at 540 nm, was also employed. Id. Both assays were validated in vitro and in living cells, and the wavelengths monitored did not overlap with absorbance characteristics of putative inhibitors.
Inhibition of N-terminal His-tagged human beta-glucuronidase expressed in Escherichia coli BL21 (DE3) assessed as residual activity using pNPG as substrate at 10 uM preincubated for 30 mins followed by substrate addition measured after 1 hr relative to control
Inhibition of beta-glucuronidase (unknown origin) using p-nitrophenyl-beta-D-glucuronide as substrate pre-incubated for 30 mins followed by substrate addition