Activity: Testosterone 6β-hydroxylase
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: 4-androstene-3,17-dione (10 µl of 10 µg/ml)
HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 40°C
Mobile phase: 0.05% orthophosphoric acid/methanol 44/56 (v/v) (isocratic separation)
Flow rate: 1.0 ml/min
Run time: 18.5 min
Injection volume: 50 µl
Detection: UV, λ = 240 nm
Retention times:
6β-hydroxytestosterone 5.5 min
4-androstene-3,17-dione 13.3 min
testosterone 17.5 min
Effect of phosphate concentration on testosterone turnover by CYP3A4R and CYP3A4LR Bactosomes:
Testosterone turnover by CYP3A4 is sensitive to the concentration of phosphate in the incubation.
These assays were carried out at 200 µM testosterone at a P450 concentration of 2.5 pmol per 200 µl.
The length of time for which product formation is linear is also affected by phosphate concentration.