Activity: Diclofenac 4′-hydroxylase
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl2
Incubation volume: 200 µl
Stop reagent: methanol (200 µl)
Column: Hypersil ODS (5 µm) 250 x 4 mm
Mobile phase: 20 mM potassium phosphate pH 7/methanol/acetonitrile 60/22.5/17.5 (v/v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 13.5 min
Injection volume: 100 µl
Detection: UV, λ = 280 nm
4′-hydroxydiclofenac 4.9 min
diclofenac 12.0 min
The metabolite, 4′-hydroxydiclofenac is available from Cypex Ltd
Effect of phosphate concentration on diclofenac turnover by CYP2C18LR Bactosomes:
Diclofenac 4′-hydroxylase activity is inhibited at phosphate concentrations above 50 mM.
This assay was carried out at 200 µM diclofenac at a P450 concentration of 5 pmol per 200 µl.