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Cypex CYP2B6
Plate Reader Assay
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.
Made
under licence from BTG International Ltd (AU730155, EP0914446,
US6566108 and other patents pending).
United States Patent Nos. 5,420,027 or 5,240,831, Canada Patent No.
2100245 and other patents pending.
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