Usefulness of statistic experimental designs in enzymology: example with recombinant hCYP3A4 and 1A2

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.