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.