The document discusses optimization of cytochrome P450 2E1 (CYP2E1) enzyme assays. Initial assays showed decreasing relative fluorescence over time, indicating a lack of enzyme activity. It was hypothesized that the wrong excitation and emission wavelengths were being used. Spectrum analysis revealed that 7-methoxy-4-trifluoromethylcoumarin (MFC) fluorescence overlapped with the product at the original wavelengths. New wavelengths of 409/530 nm reduced background noise. Kinetic studies determined the optimal MFC concentration was 11.62 μM. Acetonitrile concentration was also found to inhibit CYP2E1 activity in a dose-dependent manner.

1. 1. Hansson, T., Tindberg, N., Ingelman-Sundberg, M., Kohler, C. (1990). Regional distribution of
ethanol-inducible cytochrome p450 2E1 in the rat central nervous system. Neuroscience. 34(2), 451-
463.
2. Howard, L.A., Miksys, S., Hoffmann, E., Mash, D., Tyndale, R, F. (2003). Brain CYP2E1 is induced
by nicotine and ethanol in rat and higher in smokers and alcoholics. British Pharmacological Society.
138(7), 1376-1386.
3. Knockaert, L., Descatoire, V., Vadrot, N., Fromenty, B., Robin, M. (2011) Mitochondrial CYP2E1 is
sufficient to mediative oxidative stress and cytotoxicity induced by ethanol and acetominophen.
Toxicology in Vitro 25, 475-484.
4. Stresser, D.M., Turner, S.D., Blanchard, A.P., Miller, V.P., Crespi, C.L. (2002) Cytochrome P450
fluorometric substrates: identification of isoform selective probes for rat CYP2D2 and human
CYP3A4. Drug Metab Dispos 30, 845-852.
The Optimization of CYP2E1 Enzyme Assays
Ailin Lian, Melissa VanAlstine
Department of Chemistry, Adelphi University, Garden City, NY 11530
Discussion
.
Experimental DesignBackground Spectrum Analysis Scans
Conclusion
Cytochrome P450s (CYPs)
 Superfamily of enzymes that contain a heme, which is an iron center coordinated into
a poryphorin ring.
Enzyme assays initially showed odd results with decreasing RFU values,
represented in Figure 1. At time 0, there is a high RFU value and as time increases, the
relative fluorescence units are decreasing.
A spectrum analysis of HFC was used to set the initial excitation and emission
wavelengths of 370/500 nm, but was later hypothesized to be the wrong wavelengths to
read the enzyme assay.
A spectrum analysis of MFC was done to see whether it its fluorescence was
overlapping with the fluorescence of the product, HFC. When comparing Figure 4 and
Figure 5, Figure 5 shows that when the excitation is set at 409 nm, MFC emits RFU values
around 25 at 530 nm. Reading at the 409/530 wavelengths decreased the background noise
by over 40-fold.
Figure 6 shows that as time and concentration of MFC increases, the enzyme
activity of CYP2E1 increases linearly. Figure 7 shows that the Km value is 11.62 uM,
which means that the concentration of MFC used in the enzyme assay should be around
this number. In my enzyme assay, 70 uM MFC is used because according to the enzyme
linearity graph, using only 10 uM of MFC gives the enzyme a velocity value around 1.5,
while using 70 uM of MFC, the velocity value increases to about 2.5. The literature
turnover number for a CYP2E1 enzyme assay with 50 µM MFC is 2.23 min-1 7. The
turnover number for figure 7 at 50 uM MFC is 2.56 min-1. These values are relatively close
to one another, indicating that the experiment was done with accuracy.
Figure 8 shows that as percent acetonitrile increases, relative fluorescent units
decrease. Figure 9 illustrates that as the concentration of percent acetonitrile increases, the
more it inhibits the control. As little as 2% acetonitrile inhibits CYP2E1 by 50%.
All assays were performed using a Spectra Max Gemini XPS fluorometric plate
reader in conjunction with the computer software SoftMax Pro 5.3.
The emission and excitation scans of HFC are composed of an HFC solution
dissolved in 0.1 M Tris pH 9.0. The emission scans of MFC contains 0.5 M, pH 7.4,
potassium phosphate buffer and MFC, with final MFC concentrations ranging from 0 μM to
150 μM.
The substrate velocity curve for CYP2E1 consisted of a control, blanks for each
control, and a standard curve. The control was composed of enzyme, substrate, and the
NADPH electron regenerating system. The enzyme/substrate mix was composed of 1 uM
P450 content of CYP2E1, varied concentrations of MFC, and 0.5 M, pH 7.4, of potassium
phosphate buffer. The NADPH cofactor mix was made up of cofactors (consisting of 1.3
mM NADP+, 66 mM MgCl2, and 66 mM glucose-6-phoshate), 40 units/mL glucose-6-
phosphate-dehydrogenase, and milliQ water. The blanks used for each control were missing
the NADPH cofactor mix, so it consisted only of MFC, CYP2E1 and water. The standard
curve consisted of 0.5 M, pH 7.4, potassium phosphate buffer, 250 uM HFC and the
enzyme/substrate mix. This plate was read in 10 minute intervals starting from time 0 until
time 60, with the excitation and emission wavelengths set at 409 nm and 530 nm.
The enzyme assay that tested for the effects of percent acetonitrile on the assay also
contained the NADPH cofactor mix, the enzyme substrate mix, which was composed of 1
uM P450 content of CYP2E1 and 50 mM MFC, as well as the addition of a certain
percentage of acetonitrile in each well, ranging from 2% down to 0% acetonitrile.
The enzyme assays should be read at an excitation/emission wavelength of 409/530
nm to reduce background noise from the fluorescence of MFC. The substrate velocity
curve in conjunction with the Time vs. RFU graph shows that 70 uM MFC should be used
to obtain the most amount of enzyme activity. It was also discovered that the more
acetonitrile that is present in the CYP2E1 enzyme assay, the more CYP2E1 was inhibited,
therefore, lowering enzyme activity.
CYP2E1
 Found in the liver, brain, and central nervous system.1
 Induced by alcohol consumption and cigarette
smoking.2
 Activates procarcinogens, compounds that
become carcinogenic when undergoing metabolic
activation, and cytotoxins, which are substances
that have toxic effects on cells.1
 Metabolizes small molecules like ethanol,
acetaminophen, carbon tetrachloride and
carcinogens such as nitrosamines.3
 Transforms endogenous substrates such as acetone,
glycerol, and different fatty acids. 3
 When CYP2E1 generates large amounts of reactive oxygen species, it can result
in cellular damage. 3
 CYP2E1 reacts with its substrate 7-methoxy-4-trifluoromethylcoumarin (MFC) to
yield a fluorescent product called 7-hydroxy-4-trifluoromethylcoumarin (HFC).
0 20 40 60
0
1
2
3
4
75 uM
50 uM
25 uM
15 uM
10 uM
5 uM
2.5 uM
0 uM
Time
RFU
0 10 20 30 40 50 60 70
0
1
2
3
[MFC] uM
Velocity(min-1
)
0.0 0.5 1.0 1.5 2.0
0
2
4
6
8
% acetonitrile
RFU
0.0 0.5 1.0 1.5 2.0
0
10
20
30
40
50
60
70
80
90
100
110
% acetonitrile
%Control
7-methoxy-4-trifluoromethylcoumarin
(MFC)
7-hydroxy-4-trifluoromethylcoumarin
(HFC)
Figure 4: Emission Scan of MFC at concentrations of 150
μM, 125 μM, 100 μM, 75 μM, 50 μM, 25 μM, 10 μM, and
0 μM with the excitation wavelength set at 370 nm.
Figure 5: Emission Scan of MFC at concentrations of 150
μM, 125 μM, 100 μM, 75 μM, 50 μM, 25 μM, 10 μM, and
0 μM with the excitation wavelength set at 409 nm.
Figure 6: Time vs RFU graph of CYP2E1 read at an
excitation/emission wavelength of 409/530 nm.
Figure 7: Michaelis Menten substrate velocity curve
of CYP2E1 with 75 uM MFC at an excitation
emission wavelength of 409/530 nm at 30 minutes.
Acknowledgements
 Lendelle Raymond
 Kylie Sikorski
 Chemistry Department
Figure 8: Effects of various percentages of acetonitrile (2,
1.5, 1.25, 1.0, 0.75, 0.50, 0.25, and 0) on CYP2E1 read at
an excitation/emission wavelength of 409/530 nm at 30
minutes.
Figure 9: Percent control vs. Percent Acetonitrile for
CYP2E1 read at an excitation/emission wavelength of
409/530 nm at 30 minutes with 2, 1.50, 1.25, 1.0, 0.75,
0.50, 0.25 and 0% of acetonitrile.
Initial Results
0 20 40 60
100
125
150
175
200
225
Time
RFU
Figure 1: Time vs. RFU graph for
CYP2E1 and 70 µM MFC read at
an excitation/emission wavelength
of 370/500 nm.
What is the cause of the apparent
lack of enzyme activity?
Hypothesis
The enzyme assays are being
read at the wrong excitation and
emission wavelengths.
References
Optimization of CYP2E1 Assay Results
Vmax = 3.161± 0.2615 min-1
Km = 11.62 ± 2.932 µM
Figure 2: Emission scan of HFC with the excitation
wavelength set at 350 nm.
Figure 3: Excitation scan of HFC with the emission
wavelength set at 500 nm.
Enzymatic Activity of CYP2E1 Substrate Velocity Curve for CYP2E1
Effects of Percent Acetonitrile on CYP2E1 Percent Inhibition of CYP2E1 by Acetonitrile
Odd Results for Initial CYP2E1 Assay