Activity: Bufuralol 1′-hydroxylase
Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl2
Incubation volume: 200 µl
Stop reagent: 70% perchloric acid (10 µl)
Internal standard: none
HPLC conditions
Column: HyperClone BDS-C18 (3 µm) 125 x 4 mm
Temperature: 30°C
Mobile phase: 10 mM perchloric acid/water/acetonitrile 20/56/24 (% v/v/v) initial (gradient separation)
Flow rate: 1.0 ml/min
Run time: 12.5 min
Injection volume: 10 µl
Detection: fluorescence, λ(ex) = 252 nm; λ(em) = 302 nm
Retention times:
1′-hydroxybufuralol 2.8 min
bufuralol 5.4 min