Activity: 17β-estradiol sulfotransferase (3-sulfate formation)
Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl2, 10 mM dithiothreitol
Incubation volume: 200 µl
Stop reagent: 1 M hydrochloric acid (10 µl)
HPLC conditions
Column: HyperClone ODS (5 µm) 250 x 4 mm
Temperature: 40°C
Mobile phase: (isocratic)
41% Water
45% Acetonitrile
14% 50 mM ammonium acetate/50 mM tetrabutylammonium (pH 3.6)
Flow rate: 1.0 ml/min
Run time: 9.5 min
Injection volume: 100 µl
Detection: fluorescence λ(ex) = 211 nm, λ(em) = 312 nm
Retention times:
17β-estradiol 17-sulfate 3.9 min
17β-estradiol 3-sulfate 4.4 min
17β-estradiol 5.5 min