Usefulness of Statistic Experimental Designs in Enzymology: Example with Recombinant hCYP3A4 and 1A2

• Published in: Analytical biochemistry Vol. 276; no. 1; pp. 18 - 26
• Main Authors: Bournique, B., Petry, M., Gousset, G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.1999
• Subjects: Biometry; CYP1A2; CYP3A4; Cytochrome P-450 CYP1A2 - genetics; Cytochrome P-450 CYP1A2 - metabolism; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; drug metabolism; enzyme; experimental design; heterologous expression; Humans; In Vitro Techniques; Kinetics; Magnesium - pharmacology; membrane; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; modeling; Models, Biological; MROD; nifedipine; optimization; Osmolar Concentration; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - genetics; Substrate Specificity; yeast; enzyme; nifedipine; modeling; membrane; optimization; drug metabolism; CYP1A2; experimental design; heterologous expression; CYP3A4; MROD; yeast
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1999.4304

First, the effects of 10 incubation factors were screened altogether on nifedipine dehydrogenase (NIF) and methoxyresorufin O-deethylase (MROD) activities catalyzed by recombinant human CYP3A4 and 1A2, respectively. Using a statistic experimental design, only 36 assays were needed to be exhaustive. Eight factors influenced CYP3A4-mediated NIF activity: buffer type, pH, temperature, Mg/EDTA, cytochrome b5, NADPH-P450 reductase, NADH, and solvent. Two factors had no significant effect: total ionic concentration and catalase/reduced glutathione. Six factors influenced CYP1A2-mediated MROD rates: buffer type, pH, temperature, Mg/EDTA, NADH, and glycerol. Four factors had no significant effect: total ionic concentration, cytochrome b5, reductase, and NAD+. Secondly, the combined effects of ionic strength and Mg concentration on NIF/CYP3A4 were studied and easily modeled using another statistic experimental design. The effect of Mg was strong at a constant ionic strength of 100 mM and negligible at a constant ionic strength of 500 mM. Thirdly, the effects of influencing factors and their interactions on MROD/CYP1A2 were modeled after 40 assays using a third statistic experimental design. Later experiments confirmed the predictivity of the models and the efficiency of optimized conditions. This approach can be applied to other biochemistry areas.