Cypex CYP4F2 QC Assays

Activity: Leukotriene B₄ omega-hydroxylase

Incubation conditions
Assay buffer: 0.1 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 50 µl
Stop reagent: acetonitrile/acetic acid, 98/2 (v/v) (25 µl)
Internal standard: not used

HPLC conditions
Column: HyperClone ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: 100 mM ammonium acetate (pH 5.6)/water/methanol 20/35/45 (% v/v/v) initially (gradient separation)
Flow rate: 1.0 ml/min
Run time: 15.5 min
Injection volume: 50 µl
Detection: UV (DAD), l = 271 nm
Retention times:
      omega-hydroxy-LTB₄      10.0 min
      Leukotriene B₄               13.2 min

Effect of phosphate concentration on leukotriene B₄ turnover by CYP4F2BLR Bactosomes
Leukotriene B₄ turnover by CYP4F2 is very sensitive to the concentration of phosphate in the incubation.

This assay was carried out at 100 µM leukotriene B₄ at a P450 concentration of 2.5 pmol per 50 µl.

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.