Activity: 7-ethoxy-4-(trifluoromethyl)-coumarin (EFC) O-deethylase
General description
Incubations are set up in a black 96-well plate, and consist of substrate (EFC) and CYP2B6R or CYP2B6BR Bactosomes in phosphate buffer containing magnesium chloride. Reactions are initiated by adding 40 µl of 5x NADPH generating system [this is added to all wells, including blanks and standards]. Metabolite (HFC) formation is measured fluorometrically, using detection wavelengths chosen to minimise interference from NADPH and EFC.
The metabolite, HFC, is available from Cypex.
Important note: This test does not work with CYP2B6LR, CYP2B6LR EasyCYP, or CYP2B6R EasyCYP Bactosomes.
Incubation conditions
Plate type: black, 96-well, flat-bottomed
Assay buffer: 100 mM potassium phosphate pH 7.4, 5 mM MgCl2
Solvent used to dissolve EFC: methanol
Final EFC concentration: 2 µM
Final CYP2B6 concentration: 2 pmol/200 µl (0.5 pmol/200 µl for time linearity with CYP2B6BR)
Incubation time: 20 min
Incubation volume: 200 µl
Incubation temperature: 37°C
Metabolite standard: HFC, 8 ng/200 µl (final concentration)
Stop reagent: none (continuous monitoring)
Plate reader parameters
Detection mode: fluorescence, top reading
Excitation wavelength: 431 nm (bandwidth 9 nm)
Emission wavelength: 535 nm (bandwidth 20 nm)
Gain: calculated from HFC standard
Sampling frequency: 20 s
Time linearity test on CYP2B6BR Bactosomes with EFC as substrate:
This test was run at 2 µM EFC at a final CYP concentration of 0.5 pmol/200 µl.
The fluorescence was measured every 15 s.
The reaction is linear for approximately 25 – 30 min with CYP2B6BR Bactosomes.
Time linearity test on CYP2B6R Bactosomes with EFC as substrate:
This test was run at 2 µM EFC at a final CYP concentration of 2 pmol/200 µl.
The fluorescence was measured every 30 s.
The reaction is linear for approximately 15 – 20 min with CYP2B6R Bactosomes.
P450 linearity test on CYP2B6BR Bactosomes with EFC as substrate:
This test was run at 2 µM EFC using an incubation time of 10 min.
The fluorescence was measured every 20 s.
Activity was calculated from the maximum slope of each individual time linearity plot.
The reaction is linear up to 2 pmol/200 µl with CYP2B6BR Bactosomes.
A similar plot is seen with CYP2B6BR EasyCYP Bactosomes (not shown).
P450 linearity test on CYP2B6R Bactosomes with EFC as substrate:
This test was run at 2 µM EFC using an incubation time of 10 min.
The fluorescence was measured every 20 s.
Activity was calculated from the maximum slope of each individual time linearity plot.
The reaction is linear up to 4 pmol/200 µl with CYP2B6R Bactosomes.
Kinetics test on CYP2B6BR Bactosomes with EFC as substrate:
This test was run at 2 pmol CYP/200 µl using an incubation time of 10 min.
The fluorescence was measured every 20 s.
Activity was calculated from the maximum slope of each individual time linearity plot.
The reaction is characterised by a Vmax of ~3 pmol/min/pmol CYP and a Km of ~1.8 µM in CYP2B6BR Bactosomes. There appears to be substrate activation at EFC concentrations ≥ 10 µM.
Kinetics test on CYP2B6R Bactosomes with EFC as substrate:
This test was run at 2 pmol CYP/200 µl using an incubation time of 10 min.
The fluorescence was measured every 20 s.
Activity was calculated from the maximum slope of each individual time linearity plot.
The reaction is characterised by a Vmax of ~1.5 pmol/min/pmol CYP and a Km of ~1.8 µM in CYP2B6R Bactosomes. There appears to be substrate activation at EFC concentrations ≥ 10 µM.
We have found that this plate reader assay gives consistently lower activities than our HPLC-based assay using EFC as substrate (for example, the Vmax for CYP2B6BR is 8 – 9 pmol/min/pmol CYP by HPLC, compared with 3 – 4 pmol/min/pmol CYP on the plate reader). The reason for this difference has not been elucidated.