Activity: 7-benzyloxyquinoline (7BQ) O-debenzylase
General description
Incubations are set up in a black 96-well plate, and consist of substrate (7BQ) and CYP3A4 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 (7HQ) formation is measured fluorometrically, using detection wavelengths chosen to minimise interference from NADPH and 7BQ.
The O-debenzylation of 7BQ by CYP3A4 Bactosomes does not follow Michaelis-Menten kinetics. In addition, there is marked substrate inhibition at 7BQ concentrations >70 µM (see data below). The activity determined at 70 µM 7BQ is therefore taken to represent the maximum measurable activity, and this is used to estimate a value for S50 (substrate concentration at which the activity is 50% of the maximum measurable activity) from the experimental data. Time and P450 linearity tests are then run at a 7BQ concentration ≤ S50.
The substrate, 7BQ, and metabolite, 7HQ, are available from Cypex.
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 7BQ: methanol
Final 7BQ concentration: 13 µM (or experimentally determined S50)
Final CYP3A4 conc.: 0.5 pmol/200 µl (CYP3A4R/CYP3A4BR); 2.5 pmol/200 µl (CYP3A4LR/CYP3A4BLR)
Incubation time: 5 min
Incubation volume: 200 µl
Incubation temperature: 37°C
Metabolite standard: 7HQ, 50 ng/200 µl (final concentration)
Stop reagent: none (continuous monitoring)
Plate reader parameters
Detection mode: fluorescence, top reading
Excitation wavelength: 430 nm (bandwidth 9 nm)
Emission wavelength: 530 nm (bandwidth 20 nm)
Gain: calculated from 7HQ standard
Sampling frequency: 15 s (time linearity) or 20 s (P450 linearity or kinetics)
Time linearity test on CYP3A4R Bactosomes with 7BQ as substrate:
This test was run at 13 µM 7BQ at a final CYP concentration of 0.5 pmol/200 µl.
The fluorescence was measured every 15 s.
The reaction is linear for approximately 5 min with CYP3A4R Bactosomes.
Time linearity test on CYP3A4BR Bactosomes with 7BQ as substrate:
This test was run at 16 µM 7BQ at a final CYP concentration of 0.5 pmol/200 µl.
The fluorescence was measured every 20 s.
The reaction is linear for approximately 8 – 10 min with CYP3A4BR Bactosomes.
Time linearity test on CYP3A4LR Bactosomes with 7BQ as substrate:
This test was run at 13 µM 7BQ at a final CYP concentration of 2.5 pmol/200 µl.
The fluorescence was measured every 15 s.
The reaction is linear for approximately 10 min with CYP3A4LR Bactosomes.
Time linearity test on CYP3A4BLR Bactosomes with 7BQ as substrate:
This test was run at 15 µM 7BQ at a final CYP concentration of 2.5 pmol/200 µl.
The fluorescence was measured every 20 s.
The reaction is linear for approximately 20 min with CYP3A4BLR Bactosomes.
P450 linearity test on CYP3A4R Bactosomes with 7BQ as substrate:
This test was run at 13 µM 7BQ using an incubation time of 5 min.
The fluorescence was measured every 20 s.
The reaction is linear up to at least 2 pmol/200 µl with CYP3A4R Bactosomes. A similar plot is observed with CYP3A4BR Bactosomes (data not shown).
P450 linearity test on CYP3A4LR Bactosomes with 7BQ as substrate:
This test was run at 13 µM 7BQ using an incubation time of 5 min.
The fluorescence was measured every 20 s.
The reaction is linear up to at least 10 pmol/200 µl with CYP3A4LR Bactosomes. A similar plot is observed with CYP3A4BLR Bactosomes (data not shown).
Kinetics test on CYP3A4R Bactosomes with 7BQ as substrate:
This test was run at 0.5 pmol CYP/200 µl using an incubation time of 5 min.
The fluorescence was measured every 20 s.
The reaction is characterised by non-Michaelis-Menten kinetics in CYP3A4R Bactosomes.
7BQ concentrations >70 µM give rise to substrate inhibition. Similar plots are observed with CYP3A4BR, CYP3A4LR and CYP3A4BLR Bactosomes (data not shown).
Typical kinetic parameters for different enzyme preparations:
The activity measured at 70 µM 7BQ (80 µM for Supersomes) is used as the maximum velocity. The substrate concentration at which the activity is 50% of the maximum (S50) is estimated from the experimental data.
The maximum velocity given for Supersomes has been corrected for CYP content as measured by Cypex, using spectral analysis.