CYP1B1*3LR Bactosomes (low reductase)

CYP1B1 QC Assays

Activity: 17β-estradiol 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)
Internal standard: none

HPLC conditions
Column: HyperClone BDS-C18 (3 µm) 125 x 4 mm
Temperature: 32°C
Mobile phase: water/methanol/acetonitrile/glacial acetic acid;
48.95/34/17/0.05 (% v/v/v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 7.6 min
Injection volume: 100 µl
Detection: UV, λ = 206 nm

Retention times:
4-hydroxyestradiol 3.9 min
2-hydroxyestradiol 4.5 min
17β-estradiol 6.7 min

Effect of phosphate concentration on 17β-estradiol turnover by CYP1B1*3LR Bactosomes:

17β-estradiol turnover by CYP1B1 is sensitive to the concentration of phosphate in the incubation.

This assay was carried out at 5 µM 17β-estradiol at a P450 concentration of 5 pmol per 200 µl.

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.