Activity: 3-O-methyl-fluorescein (OMF) O-demethylase
General description
Incubations are set up in a black 96-well plate, and consist of substrate (OMF) and CYP2C19 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].
It is not possible to measure metabolite (fluorescein) formation in real time by direct fluorescence measurement (unlike other plate reader assays), for two reasons. Firstly, a more alkaline pH than that used in the incubation is required to maximise the fluorescence of the fluorescein molecule. Secondly, the fluorescence spectra of OMF and fluorescein overlap, making it very difficult to detect small amounts of fluorescein formed during an incubation.
Instead, reactions are terminated after the desired incubation time by the addition of 2 M sodium hydroxide. The resulting increase in pH maximises the fluorescence of the fluorescein molecule. More importantly, incubating the plate at 37°C for the subsequent 2 h results in the selective, NADPH-dependent degradation of residual OMF. It is important to note that the fluorescence attributable to OMF is not completely removed by this treatment – it decreases in proportion to the level present immediately before NaOH addition. However, this is sufficient to allow the fluorescein to be measured fluorometrically. It should also be noted that incubating for longer than 2 h does not result in any further significant decrease in OMF fluorescence.
As the degradation of OMF depends on the presence of NADPH in the incubation, it is very important to ensure that NADPH is added to all wells – there is no degradation in the absence of NADPH (in fact, the fluorescence signal increases). In addition, both the rate and extent of OMF degradation increase with increasing NADPH concentration, at least up to 1 mM NADPH (and possibly up to 2 mM). As a result, residual OMF is removed more thoroughly (and hence, a better signal-to-noise ratio is achieved) when the test is run using NADPH generating system, rather than NADPH, because the NADPH concentration is maintained up to the end of the initial incubation.
As a result, tests using OMF as substrate are slightly different to tests using other substrates:
- In the time linearity test, each time point corresponds to a separate well on the microplate. The fluorescence is measured across the plate only after the final sample has been incubated with 2 M NaOH for (at least) 2 h.
- In the kinetics test, each OMF concentration must have a matched control incubation containing NADPH, but no protein. This allows the residual OMF fluorescence at each OMF concentration to be measured and subtracted from the test values.
The substrate, OMF, is 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 OMF: acetonitrile
Final OMF concentration: 1 µM
Final CYP2C19 concentration: 1 pmol/200 µl (for CYP2C19R and CYP2C19LR)
Incubation time: 10 min for P450 linearity, 30 min for kinetics, 60 min for time linearity
Incubation volume: 200 µl
Incubation temperature: 37°C
Metabolite standard: fluorescein, 25 ng/200 µl (final concentration)
Stop reagent: 2 M sodium hydroxide (75 µl)
After the addition of stop reagent, the plate is incubated for 2 h at 37°C before fluorescence measurement.
Plate reader parameters
Detection mode: fluorescence, top reading
Excitation wavelength: 502 nm (bandwidth 9 nm)
Emission wavelength: 533 nm (bandwidth 20 nm)
Gain: calculated from fluorescein standard
Sampling frequency: not applicable (single reading per well)
Time linearity test on CYP2C19R Bactosomes with OMF as substrate:
This test was run at 1 µM OMF at a final CYP concentration of 1.4 pmol/200 µl.
The reaction is linear for approximately 40 min with CYP2C19R Bactosomes.
Time linearity test on CYP2C19LR Bactosomes with OMF as substrate:
This test was run at 1 µM OMF at a final CYP concentration of 1 pmol/200 µl.
The reaction is linear for at least 60 min with CYP2C19LR Bactosomes.
P450 linearity test on CYP2C19R Bactosomes with OMF as substrate:
This test was run at 1 µM OMF using an incubation time of 10 min.
The reaction is linear up to 3 pmol/200 µl with CYP2C19R Bactosomes.
P450 linearity test on CYP2C19LR Bactosomes with OMF as substrate:
This test was run at 1 µM OMF using an incubation time of 10 min.
The reaction is linear up to 3 pmol/200 µl with CYP2C19LR Bactosomes.
Kinetics test on CYP2C19R Bactosomes with OMF as substrate:
This test was run at 1 pmol CYP/200 µl using an incubation time of 30 min.
The reaction is characterised by a Vmax of 2.2 pmol/min/pmol CYP and a Km of 1.2 µM in CYP2C19R Bactosomes. OMF concentrations ≥2 µM give rise to substrate activation.
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.
