中科院西北特色植物资源化学重点实验室/甘肃省天然药物重点实验室知识库

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 (K_m) was calculated to be 1.347 mM for tyrosinase. The inhibition constant of kojic acid to free tyrosinase (K_I) and kojic acid to tyrosinase/L-DOPA complex (K_IS) 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.