CYP2B6LR Bactosomes (low reductase)

CYP2B6 QC Assays

Activity: Diazepam N-demethylase

Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: acetonitrile (100 µl)
Internal standard: none

HPLC conditions
Column: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Temperature: 30°C
Mobile phase: 200 mM potassium phosphate pH 2/Water/Acetonitrile 50/32/18 (v/v/v) (initial)
Separation: Gradient [18-32% acetonitrile from 0-7 min; hold at 32% to 8.5 min; return to 18% by 9.0 min]
Flow rate: 1.0 ml/min
Run time: 13.5 min
Injection volume: 80 µl
Detection: UV, λ = 236 nm

Retention times:
N-desmethyldiazepam 6.0 min
diazepam 9.0 min

Effect of phosphate concentration on diazepam turnover by CYP2B6LR Bactosomes:

Diazepam N-demethylation by CYP2B6 is sensitive to the concentration of phosphate in the incubation.

Activity: Bupropion 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: none

HPLC conditions
Column: HyperClone BDS-C18 (3 µm) 125 x 4 mm
Temperature: 30°C
Mobile phase: 100 mM potassium phosphate pH 2/acetonitrile/water 20/16/64 (v/v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 17.5 min
Injection volume: 100 µl
Detection: UV, λ = 196 nm

Retention times:
hydroxybupropion 6.3 min
bupropion 12.4 min

Activity: 7-ethoxy-4-(trifluoromethyl)-coumarin (7EFC) O-deethylase

Incubation conditions
Assay buffer: 0.1 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: 1 M hydrochloric acid (25 µl)
Internal standard: none

HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 35°C
Mobile phase: 0.05% orthophosphoric acid/methanol 35/65 v/v (isocratic)
Flow rate: 1.0 ml/min
Run time: 12.8 min
Injection volume: 50 µl
Detection: fluorescence λ(ex) = 324 nm, λ(em) = 458 nm

Retention times:
7-hydroxy-4-(trifluoromethyl)-coumarin 4.8 min
7-ethoxy-4-(trifluoromethyl)-coumarin 10.3 min

Effect of phosphate concentration on 7EFC turnover by CYP2B6LR Bactosomes:
7EFC O-deethylation by CYP2B6 is relatively insensitive to the concentration of phosphate in the incubation.

This assay was carried out at 20 µM 7EFC at a P450 concentration of 2 pmol per 200 µl.