Activity: Lauric acid 12-hydroxylase
Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.4, 5 mM MgCl2
Incubation volume: 100 µl
Stop reagent: acetonitrile/acetic acid, 98/2 (v/v) (50 µl)
Internal standard: not used
HPLC conditions
Column: HyperClone ODS (3 µm) 150 x 3.2 mm
Temperature: 40°C
Mobile phase: 0.05% acetic acid/acetonitrile, 65/35 (v/v) initial (gradient separation)
Flow rate: 0.6 ml/min
Run time: 17.5 min
Injection volume: 100 µl
Detection: radiometric (14C)
Retention times:
12-hydroxylauric acid 7.6 min
lauric acid 14.5 min
The substrate, lauric acid and the metabolites 11-hydroxylauric acid and 12-hydroxylauric acid are available from Cypex Ltd.