title: Photoregulation of cytochrome P450 activity by using caged compound.
abstract: Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP(+)) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP(+) resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 μm; height, 50 μm) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants.
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Midorigaoka, Ikeda 563-8577, Japan. morigaki@port.kobe-u.ac.jp
authors.forename: Kenichi
authors.initials: K
authors.lastname: Morigaki
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Mizutani
authors.forename: Emi
authors.initials: E
authors.lastname: Kanemura
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Tatsu
authors.forename: Noboru
authors.initials: N
authors.lastname: Yumoto
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Imaishi
mesh_terms: D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D019298:Glucose-6-Phosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008027:Light; D009249:NADP / Q000378:metabolism
chemical_list: D017931:DNA Primers; D009249:NADP; D019298:Glucose-6-Phosphate; D003577:Cytochrome P-450 Enzyme System