title: Kinetics and inhibition study of tyrosinase by pressure mediated microanalysis.
abstract: In the present study, pressure mediated microanalysis (PMMA), a fast, convenient and efficient capillary electrophoresis (CE) method was developed for studying enzyme kinetics of tyrosinase and inhibition kinetics of kojic acid, a model inhibitor of tyrosinase. The enzymatic reaction conditions and CE conditions were optimized in order to obtain high enzyme activity and short analysis time. By PMMA, only the product could be detected at 475 nm, and no voltage was applied to separate the product from the reaction mixture thus greatly simplifying the optimization procedure. The spectrophotometric assay and electrophoretically mediated microanalysis (EMMA) were also performed to validate the developed method. With the present method, the Michaelis-Menten constant (Km) was calculated to be 1.347 mM for tyrosinase. The inhibition constant of kojic acid to free tyrosinase (KI) and kojic acid to tyrosinase/L-DOPA complex (KIS) were calculated to be 36.64 and 74.35 μM, respectively, and the half-maximal inhibitory concentration (IC50) was determined to be 46.64 μM for kojic acid. The developed method is fast and convenient for studying enzyme kinetics, inhibition kinetics and further screening enzyme inhibitors.
authors.affiliation: Key Laboratory of Chemistry of Northwestern Plant Resources, Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China; University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, PR China.
authors.forename: Dong-Mei
authors.initials: DM
authors.lastname: Liu
authors.affiliation: Key Laboratory of Chemistry of Northwestern Plant Resources, Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China.
authors.forename: Jun-Li
authors.initials: JL
authors.lastname: Yang
authors.affiliation: Key Laboratory of Chemistry of Northwestern Plant Resources, Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ha
authors.affiliation: Key Laboratory of Chemistry of Northwestern Plant Resources, Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China. Electronic address: chenjuan@licp.cas.cn.
authors.forename: Juan
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Chemistry of Northwestern Plant Resources, Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China. Electronic address: shiyp@licp.cas.cn.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Shi
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors*; D011312:Pressure; D011753:Pyrones / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D011753:Pyrones; C011890:kojic acid; D014442:Monophenol Monooxygenase
keywords: Enzyme kinetics study; Inhibition study; Kojic acid; Pressure mediated microanalysis; Tyrosinase