Cytochrome P450 Protocols (3^rd Ed., 3rd ed. 2013) Methods in Molecular Biology Series, Vol. 987

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.

Bioluminescent Assays for Cytochrome P450 Enzymes. In Vitro Drug-Drug Interaction Assay.- High-Throughput Mass Spectrometric Cytochrome P450 Inhibition Screening.- The Synthesis, Characterization and Application of ¹³C-Methyl Isocyanide as an NMR Probe of Heme Protein Active Sites.- High-Throughput Fluorescence Assay for Cytochrome P450 Mechanism-Based Inactivators.- Identification of Endogenous Substrates of Orphan Cytochrome P450 Enzymes Through the Use of Untargeted Metabolomics Approaches.- Genetic and Mass Spectrometric Tools for Elucidating the Physiological Function(s) of Cytochrome P450 Enzymes from Mycobacterium Tuberculosis.- An Escherichia coli Expression-Based Approach for Porphyrin Substitution in Heme Proteins.- Expression in Escherichia coli of a Cytochrome P450 Enzyme with a Cobalt Protoporphyrin IX Prosthetic Group.- Nanodiscs in the Studies of Membrane-Bound Cytochrome P450 Enzymes.- Rapid LC-MS Drug Metabolite Profiling Using Bioreactor Particles.- Fluorescence-Based Screening of cytochrome P450 activities in Intact Cells.- Screening for Cytochrome P450 Reactivity with a Reporter Enzyme.- High-Throughput Fluorescence Assay of Cytochrome P450 3A4.- Targeted Protein Capture for Analysis of Electrophile-Protein Adducts.- DNA Shuffling of Cytochrome P450 Enzymes.- Measurement of P450 Difference Spectra Using Intact Cells.- DNA Shuffling of Cytochromes P450 for Indigoid Pigment Production.- P450 oxidoreductase – Genotyping, Expression, Purification of Recombinant Protein and Activity Assessments of Wild-Type and Mutant Protein.- LICRED: A Versatile Drop-In Vector for Rapid Generation of Redox-Self-Sufficient Cytochromes P450.- Update on Allele Nomenclature for Human Cytochromes P450 and the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Database.- Simultaneous in vivo Phenotyping of CYP Enzymes.- Detection of Regulatory Polymorphisms: High-Throughput Capillary DNase-I Footprinting.- Isolation of MouseHepatocytes.- Highly Efficient SiRNA and Gene Transfer into Hepatocyte-like HepaRG Cells and Primary Human Hepatocytes: New Means for Drug Metabolism and Toxicity Studies.