CYP2C9BHR Bactosomes

CYP2C9 QC Assays

Activity: Diclofenac 4′-hydroxylase

Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: methanol (200 µl)

HPLC conditions
Column: HyperClone ODS (5 µm) 250 x 4 mm
Temperature: 30°C
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

Retention times:
4′-hydroxydiclofenac 4.9 min
diclofenac 12.0 min

The CYP2C9 substrate diclofenac and metabolite, 4′-hydroxydiclofenac, are available from Cypex.

Activity: Tolbutamide 4-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)

HPLC conditions
Column: HyperClone ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: Water/100 mM potassium phosphate pH 2/methanol/acetonitrile, 50/10/35/5 (v/v/v/v) (initial conditions)
Run type: gradient (5 – 25% acetonitrile over 4 min)
Flow rate: 1.0 ml/min
Run time: 16 min
Injection volume: 100 µl
Detection: UV, λ = 230 nm

Retention times:
4-hydroxytolbutamide 5.9 min
(carboxytolbutamide 6.8 min)
tolbutamide 9.1 min

The CYP2C9 substrate tolbutamide and metabolite 4-hydroxytolbutamide are available from Cypex.

Effect of phosphate concentration on diclofenac turnover by CYP2C9HR Bactosomes:

Diclofenac 4′-hydroxylase activity is inhibited at phosphate concentrations above 100 mM.

This assay was carried out at 50 µM diclofenac at a P450 concentration of 0.4 pmol per 200 µl.