Activity: 7-ethoxycoumarin O-deethylase
Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl2
Incubation volume: 200 µl
Stop reagent: 1 M hydrochloric acid (20 µl)
Internal standard: 4-methylumbelliferone (10 µl of 0.1 µg/ml)
HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: 0.05% orthophosphoric acid/acetonitrile (gradient separation)
Flow rate: 1.0 ml/min
Run time: 14.5 min
Injection volume: 50 µl
Detection: fluorescence λ(ex) = 324 nm, λ(em) = 458 nm
Retention times:
umbelliferone: 5.2 min
4-methylumbelliferone: 7.0 min
7-ethoxycoumarin: 10.3 min
Activity: 7-ethoxyresorufin O-deethylase
Incubation conditions
Assay buffer: 0.1 M potassium phosphate pH 7.4, 5 mM MgCl2
Incubation volume: 200 µl
Stop reagent: methanol (100 µl)
Internal standard: none
HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 35°C
Mobile phase: 20 mM potassium phosphate pH 7/methanol 58/42 (v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 5.5 min
Injection volume: 75 µl
Detection: fluorescence λ(ex) = 530 nm, λ(em) = 580 nm
Retention times:
resorufin: 4.0 min
The substrate 7-ethoxyresorufin and the metabolite resorufin are both available from Cypex Ltd.