Activity: Chlorzoxazone 6-hydroxylase
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: zoxazolamine (10 µl of 100 µg/ml)
Column: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Mobile phase: 0.05% orthophosphoric acid/acetonitrile 81/19 (v/v) (isocratic separation)
Flow rate: 1.0 ml/min
Run time: 10.8 min
Injection volume: 50 µl
Detection: UV, λ = 295 nm
6-hydroxychlorzoxazone 2.3 min
Zoxazolamine 5.1 min
Chlorzoxazone 10.0 min
Effect of phosphate concentration on chlorzoxazone turnover by CYP2E1R Bactosomes:
Chlorzoxazone turnover by CYP2E1 is sensitive to the concentration of phosphate in the incubation.
This assay was carried out at 500 µM chlorzoxazone at a P450 concentration of 10 pmol per 200 µl.
The length of time for which product formation is linear is also affected by phosphate concentration.