Activity: Leukotriene B4 ω-hydroxylase
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
Column: HyperClone ODS (5 µm) 250 x 4 mm
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), λ = 271 nm
ω-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.