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hCYP1A2 (reductase) |
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Description:
Recombinant human cytochrome P4501A2 (hCYP1A2) has a broad array of applications ranging from the evaluation of potential pharmaceutical agents to the catalysis of synthetic reactions. Recombinant hCYP1A2 is useful for high throughput screening of drug candidates for possible toxicity, drug-drug interaction and dosage issues. Comparisons of the bacterially expressed human CYP1A2 with human liver microsomes give similar kinetic profiles (1). The supplied hCYP1A2 has a 11 amino acid deletion and an S18V, A19F and I20L changes in the N-terminal membrane anchor region that improves expression in E. coli but has not been found to alter enzyme activity (2). Human NADPH-cytochrome P450 reductase is also present to permit efficient hCYP1A2 activity.
Source:
The membrane fraction from E. coli DH5α-T1r co-expressing human CYP1A2 and human NADPH-cytochrome P450 reductase.
Enzyme Properties
Heat Inactivation:
No
Specific Activity:
3.7 pmol
Concentration
P450:
1 μM
Total Protein: 0.9 mg/ml
Storage Conditions
Storage Conditions:
100 mM KPO4
pH 7.4 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
Usage notes:- Upon receiving, aliquot and store at -70°C to avoid multiple freeze-thaw cycles.
For use thaw in 37°C water bath and immediately place on ice. - This enzyme was produced using the Pharmazyme™ technology licensed to NEB by PPD Discovery Inc.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
hCYP1A2 Assay Conditions::
hCYP1A2 activity was evaluated by following its conversion of phenacetin to 4-acetamidophenol. The reaction buffer was 50 mM potassium phosphate, (pH 7.5) containing 3.3 mM glucose-6-phosphate, 0.4 units/ml glucose-6-phosphate dehydrogenase, 3.3 mM MgCl2, 1.3 mM NADP+ and 200 µM phenacetin. The addition of hCYP1A2 to a final concentration of 40 nM initiated the assay. At specific time points the reaction was quenched by adding acetonitrile (at 10% of the reaction volume). Formation of 4-acetamidophenol was followed by HPLC using a C18 column and a 50 mM potassium phosphate (pH 7.4) buffer containing 10% acetonitrile under isocratic conditions. Products and reactants were detected by monitoring their absorbance at 254 nm. The amount of product was determined by comparison to a standard of 4-acetamidophenol.
References
- Parikh, A. et al. (1997) Nature Biotech., 15, 784-788.
- Sandhu, P. et al. (1994) Archives Biochem. Biophys., 309, 168-177.
- Barnes, H.J. (1996) E. Johnson and M. Waterman (Eds.), Methods Enzymol., 272, pp. 3-14. San Diego: Academic Press.
- Guengerich, F.P. et al. (1996) E. Johnson and M. Waterman (Eds.), Methods Enzymol., 272, pp. 35-45. San Diego: Academic Press.
Companion Products
E. coli DH5α Control Membranes
hCYP2C19 (reductase)
hCYP2C8 (reductase)
hCYP2D6 (reductase)
hCYP3A4(reductase)
Legal
Licenses/Patents/Disclaimers:
This Product is a Pharmazyme™ isozymes, produced underlicense from PPD Discovery, Inc. (U.S. Patent Nos. 5,240,831 and 5,420,027). Pharmazyme™ is a trademark of PPD, Inc. This product is sold for research use only and not for resale in any form. Commercial use of these products may require a license from PPD Discovery, Inc., Morrisville, NC.
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