Cypex CYP3A5 QC Assays

Activity: Testosterone 6b-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: 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, l = 240 nm
Retention times:
      6b-hydroxytestosterone      5.5 min
      4-androstene-3,17-dione   13.3 min
      testosterone                     17.5 min

Effect of phosphate concentration on testosterone turnover by CYP3A5LR Bactosomes
Testosterone turnover by CYP3A5 is sensitive to the concentration of phosphate in the incubation.

This assay was carried out at 320 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.

Made under licence from BTG International Ltd (AU730155, EP0914446, US6566108 and other patents pending).

United States Patent Nos. 5,420,027 or 5,240,831, Canada Patent No. 2100245 and other patents pending.