Substrate: 1-naphthol
Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.5 (at 37°C), 5 mM MgCl2, 10 µg/ml alamethicin (for kinetics only)
Incubation volume: 200 µl
Stop reagent: acetonitrile (100 µl)
HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: 2% acetic acid/water/acetonitrile, 40/40/20 (v/v/v) (initial)
Separation type: Gradient
Flow rate: 1.0 ml/min
Run time: 13 min
Injection volume: 20 µl
Detection: fluorescence λ(ex) = 280 nm, λ(em) = 335 nm
Retention times:
1-naphthylglucuronide 5.4 min
1-naphthol 7.9 min
Substrate: 4-methylumbelliferone (4-MU)
Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.5 (at 37°C), 5 mM MgCl2, 10 µg/ml alamethicin (for kinetics only)
Incubation volume: 200 µl (time/protein linearity) or 1000 µl (kinetics)
Stop reagent: 1 M hydrochloric acid (20 µl for time/protein linearity; 100 µl for kinetics)
HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: 0.05% orthophosphoric acid/acetonitrile (gradient separation)
Flow rate: 1.0 ml/min
Run time: 15.0 min
Injection volume: 10 µl
Detection: fluorescence λ(ex) = 315 nm, λ(em) = 375 nm
Retention times:
4-MU glucuronide 3.8 min
4-methylumbelliferone 8.9 min