Activity: Phthalazine oxidase
Incubation conditions
Assay buffer: 0.025 M potassium phosphate pH 7.4, 0.1 mM EDTA
Incubation volume: 100 µl
Stop reagent: 1 M hydrochloric acid (12.5 µl)
HPLC conditions
Column: Gemini C18 (3 µm) 100 x 4.6 mm
Temperature: 20°C
Mobile phase: 0.05% (v/v) orthophosphoric acid/acetonitrile; 85/15 (v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 7.0 min
Injection volume: 50 µl
Detection: fluorescence λ(ex) = 285 nm, λ(em) = 340 nm
Retention times:
phthalazine 1.6 min
1-phthalazinone 4.5 min