Cypex CYP2C9 Plate Reader Assay

Activity:  7-methoxy-4-(trifluoromethyl)-coumarin (MFC) O-demethylase

General description
Incubations are set up in a black 96-well plate, and consist of substrate (MFC) and CYP2C9HR 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 MFC.

The substrate, MFC, and metabolite, HFC, are both available from Cypex.

Important note: This test does not work with 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 MFC: acetonitrile (do not use methanol)
Final MFC concentration: 40 µM
Final CYP2C9 concentration: 5 pmol/200 µl
Incubation time: 30 min
Incubation volume: 200 µl
Incubation temperature: 37°C
Metabolite standard: HFC, 50 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: 30 s

Time linearity test on CYP2C9HR Bactosomes with MFC as substrate:
This test was run at 40 µM MFC at a final CYP concentration of 5 pmol/200 µl.
The fluorescence was measured every 30 s.

There is a time lag of 5 - 15 min before maximal activity is reached. This is also seen with Supersomes. The time lag is more pronounced at higher substrate concentrations, and less pronounced at lower substrate concentrations. The reaction is then linear for the next 15 - 20 min.

Time linearity test on CYP2C9R Bactosomes with MFC as substrate:
This test was run at 40 µM MFC at a final CYP concentration of 5 pmol/200 µl.
The fluorescence was measured every 30 s.

There is a time lag of 5 - 15 min before maximal activity is reached. This is also seen with Supersomes. The time lag is more pronounced at higher substrate concentrations, and less pronounced at lower substrate concentrations. The reaction is then linear for the next 15 - 20 min.

P450 linearity test on CYP2C9HR Bactosomes with MFC as substrate:
This test was run at 40 µM MFC using an incubation time of 30 min.
The fluorescence was measured every 30 s. Activity was calculated from the maximum slope of each individual time linearity plot (after the time lag, see above).

The reaction is linear up to 8 pmol/200 µl with CYP2C9HR Bactosomes. A similar plot is seen with CYP2C9BHR Bactosomes (not shown).

P450 linearity test on CYP2C9R Bactosomes with MFC as substrate:
This test was run at 40 µM MFC using an incubation time of 30 min.
The fluorescence was measured every 30 s. Activity was calculated from the maximum slope of each individual time linearity plot (after the time lag, see above).

The reaction is only linear up to 3 pmol/200 µl with CYP2C9R Bactosomes. Therefore, activities determined at 5 pmol CYP/200 µl (the concentration we recommend) will be slightly under-estimated. Similar plots are seen with CYP2C9BR and CYP2C9*2R Bactosomes (not shown).

Kinetics test on CYP2C9HR Bactosomes with MFC as substrate:
This test was run at 5 pmol CYP/200 µl using an incubation time of 30 min.
The fluorescence was measured every 30 s. Activity was calculated from the maximum slope of each individual time linearity plot (after the time lag, see above).

The reaction is characterised by a Vmax of 2.1 pmol/min/pmol CYP and a Km of ~40 µM in CYP2C9HR Bactosomes.

Kinetics test on CYP2C9R Bactosomes with MFC as substrate:
This test was run at 5 pmol CYP/200 µl using an incubation time of 30 min.
The fluorescence was measured every 30 s. Activity was calculated from the maximum slope of each individual time linearity plot (after the time lag, see above).

The reaction is characterised by a Vmax of 0.4 pmol/min/pmol CYP and a Km of ~40 µM in CYP2C9R Bactosomes.

Kinetics test on CYP2C9*2R Bactosomes with MFC as substrate:
This test was run at 5 pmol CYP/200 µl using an incubation time of 30 min.
The fluorescence was measured every 30 s. Activity was calculated from the maximum slope of each individual time linearity plot (after the time lag, see above).

The reaction is characterised by a Vmax of 0.6 - 0.8 pmol/min/pmol CYP and a Km of 50 µM in CYP2C9*2R Bactosomes.

Kinetics test on CYP2C9*3R Bactosomes with MFC as substrate:
This test was run at 5 pmol CYP/200 µl using an incubation time of 30 min.
The fluorescence was measured every 30 s. Activity was calculated from the maximum slope of each individual time linearity plot (after the time lag, see above).

The reaction is characterised by a Vmax of <0.1 pmol/min/pmol CYP and a Km of 37 - 63 µM in CYP2C9*3R 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.

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.