Cypex CYP2E1 QC Assays

Activity: Chlorzoxazone 6-hydroxylase

Incubation conditions
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)

HPLC conditions
Column: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Temperature: 30°C
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, l = 295 nm
Retention times:
      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.

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.