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 AssaysAilin Lian, Melissa VanAlstine

Department of Chemistry, Adelphi University, Garden City, NY 11530

Discussion

.

Experimental Design Background 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 40 600

1

2

3

4 75 uM50 uM25 uM15 uM10 uM5 uM2.5 uM0 uM

Time

RFU

0 10 20 30 40 50 60 700

1

2

3

[MFC] uM

Velo

city

(min-1)

0.0 0.5 1.0 1.5 2.00

2

4

6

8

% acetonitrile

RFU

0.0 0.5 1.0 1.5 2.00

102030405060708090

100110

% acetonitrile

% C

ontro

l

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 60100

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?

HypothesisThe 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