Activity: Coumarin 7-hydroxylase
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 75/25 (v/v) (isocratic separation)
Flow rate: 1.0 ml/min
Run time: 12.5 min
Injection volume: 50 µl
Detection: fluorescence λ(ex) = 324 nm, λ(em) = 458 nm
Retention times:
umbelliferone 5.2 min
4-methylumbelliferone 7.2 min
coumarin 10.1 min