Cytochromes P450 (CYPs) comprise a large superfamily of proteins that are of central importance in the detoxification or activation of a tremendous number of natural and synthetic hydrophobic xenobiotics, including many therapeutic drugs, chemical carcinogens and environmental pollutants. CYPs are important in mediating interactions between an organism and its chemical environment and in the regulation of physiological processes. Cytochrome P450 Protocols, Third Edition focuses on high-throughput methods for the simultaneous analysis of multiple CYPs, substrates or ligands. Although the emphasis is on CYPs of mammalian origin, it reflects an increasing interest in CYPs of bacterial species. Also included are chapters on cytochrome P450 reductase (the redox partner of CYPs) and the flavin-containing monooxygenases (FMOs), and metabolomic and lipidomic approaches for identification of endogenous substrates of CYPs (¿de-orphanizing¿ CYP substrates). Written in the successful Methods in Molecular Biology¿ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.
 
Authoritative and easily accessible, Cytochrome P450 Protocols, Third Edition provides a wide range of techniques accessible to researchers in fields as diverse as biochemistry, molecular biology, pharmacology, toxicology, environmental biology and genetics.

1. Bioluminescent Assays for Cytochrome P450 Enzymes            Douglas S. Auld, Henrike Veith, and James J. Cali 2. In Vitro Drug-Drug Interaction Assay                   Michael Zientek and Kuresh Youdim 3. High-Throughput Mass Spectrometric Cytochrome P450 Inhibition Screening            Kheng B. Lim, Can C. Ozbal, and Daniel B. Kassel 4. The Synthesis, Characterization and Application of 13C-Methyl Isocyanide as an NMR Probe of Heme Protein Active Sites            Christopher McCullough, Phani Kumar Pullela, Sang-Choul Im, Lucy Waskell, Daniel Sem 5. High-Throughput Fluorescence Assay for Cytochrome P450 Mechanism-Based Inactivators                 Cesar Kenaan, Haoming Zhang, and Paul F. Hollenberg 6. Identification of Endogenous Substrates of Orphan Cytochrome P450 Enzymes Through the Use of Untargeted Metabolomics Approaches            Qian Cheng and F. Peter. Guengerich 7. Genetic and Mass Spectrometric Tools for Elucidating the Physiological Function(s) of Cytochrome P450 Enzymes from Mycobacterium Tuberculosis            Hugues Ouellet, Eric D. Chow, Shenheng Guan, Jeffery S. Cox, Alma L. Burlingame, and Paul R. Ortiz de Montellano 8. An Escherichia coli Expression-Based Approach for Porphyrin Substitution in Heme Proteins               Michael B. Winter, Joshua J. Woodward, and Michael A. Marletta 9. Expression in Escherichia coli of a Cytochrome P450 Enzyme with a Cobalt Protoporphyrin IX Prosthetic Group            Wesley E. Straub, Clinton R. Nishida, and Paul R. Ortiz de Montellano 10. Nanodiscs in the Studies of Membrane-Bound Cytochrome P450 Enzymes                  A. Luthra, M. Gregory, Y.V. Grinkova, I.G. Denisov , and S.G. Sligar 11. Rapid LC-MS Drug Metabolite Profiling Using Bioreactor Particles            Linlin Zhao, Besnik Bajrami, and James F. Rusling 12. Fluorescence-Based Screening of cytochrome P450 activities in Intact Cells            M. Teresa Donato and M. José Gómez-Lechón 13. Screening for Cytochrome P450 Reactivity with a Reporter Enzyme            Kersten S. Rabe and Christof M. Niemeyer 14. High-Throughput Fluorescence Assay of Cytochrome P450 3A4                       Qian Cheng and F. Peter. Guengerich 15. Targeted Protein Capture for Analysis of Electrophile-Protein Adducts            Rebecca E. Connor, Simona G. Codreanu, Lawrence J. Marnett, and Daniel C. Liebler 16. DNA Shuffling of Cytochrome P450 Enzymes            James B.Y.H. Behrendorff, Wayne A. Johnston, and Elizabeth M.J. Gillam 17. Measurement of P450 Difference Spectra Using Intact Cells            Wayne A. Johnston and Elizabeth M.J. Gillam 18. DNA Shuffling of Cytochromes P450 for Indigoid Pigment Production            Nedeljka N. Rosic 19. P450 oxidoreductase ¿ Genotyping, Expression, Purification of Recombinant Protein and Activity Assessments of Wild-Type and Mutant Protein            Vishal Agrawal and Walter L. Miller 20. LICRED: A Versatile Drop-In Vector for Rapid Generation of Redox-Self-Sufficient Cytochromes P450            Federico Sabbadin, Gideon G. Grogan and Neil C. Bruce 21. Update on Allele Nomenclature for Human Cytochromes P450 and the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Database            Sarah C. Sim and Magnus Ingelman-Sundberg 22. Simultaneous in vivo Phenotyping of CYP Enzymes            Sussan Ghassabian and Michael Murray 23. Detection of Regulatory Polymorphisms: High-Throughput Capillary DNase-I Footprinting            Matthew Hancock and Elizabeth A. Shephard 24. Isolation of Mouse Hepatocytes            Mina Edwards, Lyndsey Houseman, Ian R. Phillips, and Elizabeth A. Shephard 25. Highly Efficient SiRNA and Gene Transfer into Hepatocyte-like HepaRG Cells and Primary Human Hepatocytes: New Means for Drug Metabolism and Toxicity Studies            Véronique Laurent, Denise Glaise, Tobias Nübel, David Gilot, Anne Corlu, and Pascal Loyer