Activity: Paclitaxel 6α-hydroxylase
Assay buffer: 0.1 M potassium phosphate pH 7.4, 5 mM MgCl2
Incubation volume: 200 µl
Stop reagent: acetonitrile (100 µl)
Column: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Mobile phase: 20 mM potassium phosphate pH 7/acetonitrile 57.5/42.5 (v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 8.5 min
Injection volume: 75 µl
Detection: UV, λ = 230 nm
6α-hydroxypaclitaxel 4.5 min
paclitaxel 18.5 min
The substrate, paclitaxel, and metabolite, 6α-hydroxypaclitaxel are both available from Cypex Ltd.
Effect of phosphate concentration on paclitaxel turnover by CYP2C8R Bactosomes:
Paclitaxel 6α-hydroxylation by CYP2C8 is sensitive to the concentration of phosphate in the incubation.
This assay was carried out at 25 µM paclitaxel at a P450 concentration of 2 pmol per 200 µl.