|
Cypex CYP1A2
Plate Reader Assay
Activity:
7-ethoxyresorufin (7ER) O-deethylase
General description
Resorufin and 7-ethoxyresorufin are light sensitive. We
therefore recommend carrying out this procedure under yellow light, in
order to protect the integrity of the stock solutions. Incubations are
set up in a black 96-well plate, and consist
of substrate (7ER) and CYP1A2 Bactosomes in phosphate buffer
containing magnesium chloride. Reactions are initiated by adding 40
µl of 5x NADPH generating system [this can be omitted from
wells containing blanks and standards]. Metabolite
(resorufin)
formation is measured fluorometrically, using detection wavelengths
chosen to minimise interference from NADPH and 7ER.
The substrate, 7-ethoxyresorufin,
and
metabolite, resorufin, are
both
available from Cypex.
Incubation
conditions
Plate type: black, 96-well, polypropylene (polystyrene acceptable)
Assay buffer: 100 mM potassium phosphate pH 7.4, 5
mM MgCl2
Solvent used to dissolve 7ER: dimethyl sulfoxide (DMSO)
Final 7ER concentration: 0.3 µM (for CYP1A2R and
CYP1A2LR Bactosomes; can be higher for EasyCYP)
Final CYP1A2 concentration (time linearity test):
0.4
pmol/200
µl (CYP1A2R, including EasyCYP); 1.0
pmol/200 µl (CYP1A2LR, including EasyCYP)
Final CYP1A2 concentration (kinetics test):
1.0
pmol/200
µl (CYP1A2R, including EasyCYP); 2.5
pmol/200 µl (CYP1A2LR, including EasyCYP)
Incubation time: 10 min (can be longer if looking for extended time
linearity)
Incubation volume: 200 µl
Incubation temperature: 37°C
Metabolite standard: resorufin, 1 ng/200 µl (final
concentration)
Stop reagent: none (continuous monitoring)
This assay is also suitable for CYP1A2R EasyCYP Bactosomes and CYP1A2LR
EasyCYP Bactosomes. Note that P450 linearity tests on these
products must be run at a constant total protein concentration - this
is achieved by adding Control (EasyCYP) Bactosomes to incubations.
Plate
reader parameters
Detection
mode: fluorescence, top reading
Excitation wavelength: 572 nm (bandwidth 9 nm)
Emission wavelength: 604 nm (bandwidth 20 nm)
Gain: calculated from resorufin standard
Sampling frequency: 30 s
Note:
Unchanged 7-ethoxyresorufin (with fluorescence maxima at 490 nm
excitation and 580 nm emission) interferes with resorufin quantitation
at the detection wavelengths commonly used for this assay (530 nm
excitation and 590 nm emission). The wavelengths we use (572/604 nm)
keep this interference to a minimum. Furthermore, NADPH
fluorescence at these wavelengths is minimal, allowing NADPH to be
omitted from blanks and standards if desired.
Time
linearity test on CYP1A2R
Bactosomes with 7ER as substrate:
This
test was run at 0.3 µM 7ER at a final CYP concentration of
0.4
pmol/200 µl.
The fluorescence was measured every 30 s.
The
reaction is linear for approximately 8 min with CYP1A2R Bactosomes. A
similar plot is seen wth CYP1A2R EasyCYP Bactosomes, although the
linearity is sometimes a little better (up to 10 min; not shown).
Time
linearity test on CYP1A2LR
Bactosomes with 7ER as substrate:
This
test was run at 0.3 µM 7ER at a final CYP concentration of
1.0
pmol/200 µl.
The fluorescence was measured every 30 s.
The
reaction is linear for approximately 15 min with CYP1A2LR Bactosomes. A
similar plot is seen with CYP1A2LR EasyCYP Bactosomes, although the
linearity is sometimes a little better (up to 20 - 25 min; not
shown).
P450
linearity test on CYP1A2R
Bactosomes with 7ER as substrate:
This
test was run at 0.3 µM 7ER using an incubation time of 10
min.
The fluorescence was measured every 30 s.
The
reaction is linear up to (at least) 4 pmol/200 µl with
CYP1A2R
Bactosomes.
P450
linearity test on CYP1A2R
EasyCYP Bactosomes with 7ER as substrate:
This
test was run at 0.3 µM 7ER using an incubation time of 10 min.
Control EasyCYP Bactosomes were added to give a protein
concentration of 40 µg/200 µl (0.2 mg/ml) in all
wells. Controlling the total protein concentration is necessary to
obtain a linear response in this test.
The fluorescence was measured every 30 s.
The
reaction is linear up to (at least) 4 pmol/200 µl with
CYP1A2R
EasyCYP Bactosomes.
Kinetics
test on CYP1A2R
Bactosomes with 7ER as substrate:
This
test was run at 1.0 pmol CYP/200 µl using an
incubation time of 10 min.
The fluorescence was measured every 30 s.
The
reaction is characterised by a Vmax of 1.0 pmol/min/pmol CYP and a Km
of 0.3 µM in CYP1A2R Bactosomes.
Typical
kinetic parameters for different enzyme preparations:
Kinetic
parameters were estimated by fitting the experimental data directly to
the Michaelis-Menten equation using Microsoft Excel.
The
apparent Km for this reaction increases with increasing protein
concentration - this can be seen by comparing the Km in the above table
for EasyCYP and non-EasyCYP versions of the same product. The effect is
only noticeable at protein concentrations greater than about 4
µg/200 µl (0.02 mg/ml; data not shown). This
explains the need to control the total protein concentration in P450
linearity tests on EasyCYP Bactosomes, where the total protein
concentration is higher (see above), whereas this is not necessary for
tests on non-EasyCYP Bactosomes.
We have found that this plate reader assay gives consistently lower
activities than our HPLC-based assay using 7ER as substrate (for
example, the Vmax for CYP1A2R is 4
pmol/min/pmol CYP by HPLC, compared with 1 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.
|
