Cytochrome P450 protocols

Preface . . v
Contributors . . ix

1 Bioluminescent Assays for Cytochrome P450 Enzymes . . 1
2 Simultaneous Determination of Multiple GYP Inhibition Constants using a Cocktail-Probe Approach . . 11
3 High-Throughput Mass Spectrometric Cytochrome P450 Inhibition Screening . . 25
4 The Synthesis, Characterization, and Application of 13C-Methyl Isocyanide as an NMR Probe of Heme Protein Active Sites . . 51
5 High-Throughput Fluorescence Assay for Cytochrome P450 Mechanism-Based Inactivators . . 61
6 Identification of Endogenous Substrates of Orphan Cytochrome P450 Enzymes Through the Use of Untargeted Metabolomics Approaches . . 71
7 Genetic and Mass Spectrometric Tools for Elucidating the Physiological Function(s) of Cytochrome P450 Enzymes from Mycobacterium tuberculosis . . 79
8 An Escherichia coli Expression-Based Approach for Porphyrin Substitution in Heme Proteins . . 95
9 Expression in Escherichia coli of a Cytochrome P450 Enzyme with a Cobalt Protoporphyrin IX Prosthetic Group . . 107
10 Nanodiscs in the Studies of Membrane-Bound Cytochrome P450 Enzymes . . 115
11 Rapid LC-MS Drug Metabolite Profiling Using Bioreactor Particles . . 129
12 Fluorescence-Based Screening of Cytochrome P450 Activities in Intact Cells . . 135
13 Screening for Cytochrome P450 Reactivity with a Reporter Enzyme . . 149
14 High-Throughput Fluorescence Assay of Cytochrome P450 3A4 . . 157
15 Targeted Protein Capture for Analysis of Electrophile-Protem Adducts . . 163
16 DNA Shuffling of Cytochrome P450 Enzymes . . 177
17 Measurement of P450 Difference Spectra Using Intact Cells . . 189
18 DNA Shuffling of Cytochromes P450 for Indigoid Pigment Production . . 205
19 P450 Oxidoreductase: Genotyping, Expression, Purification of Recombinant Protein, and Activity Assessments of Wild-Type and Mutant Protein . . 225
20 LICKED: A Versatile Drop-In Vector for Rapid Generation of Redox-Sclf-Sufficient Cytochromcs P450 . . 239
21 Update on Allele Nomenclature for Human Cytochromes P450 and the Human Cytochrome P450 Allele (CYP-Allele) Nomenclature Database . . 251
22 Simultaneous In Vivo Phenotyping of CYP Enzymes . . 261
23 Detection of Regulatory Polymorphisms: High-Throughput Capillary DNase I Footprinting . . 269
24 Isolation of Mouse Hepatocytes . . 283
25 Highly Efficient SiRNA and Gene Transfer into Hepatocyte-Like HepaRG Cells and Primary Human Hepatocytes: New Means for Drug Metabolism and Toxicity Studies . . 295

Index . . 315