Liu Dong-Mei, Yang Jun-Li, Ha Wei, Chen Juan, Shi Yan-Ping
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.
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.
Anal Biochem. 2017 May 15;525:54-59. doi: 10.1016/j.ab.2017.02.020. Epub 2017 Mar 1.
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) was calculated to be 1.347 mM for tyrosinase. The inhibition constant of kojic acid to free tyrosinase (K) and kojic acid to tyrosinase/L-DOPA complex (K) were calculated to be 36.64 and 74.35 μM, respectively, and the half-maximal inhibitory concentration (IC) 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.
在本研究中，开发了一种压力介导的微分析方法（PMMA），这是一种快速、便捷且高效的毛细管电泳（CE）方法，用于研究酪氨酸酶的酶动力学以及曲酸（酪氨酸酶的一种模型抑制剂）的抑制动力学。对酶促反应条件和毛细管电泳条件进行了优化，以获得高酶活性和短分析时间。通过压力介导的微分析方法，仅能在475 nm处检测到产物，并且无需施加电压来从反应混合物中分离产物，从而极大地简化了优化过程。还进行了分光光度法测定和电泳介导的微分析（EMMA）以验证所开发的方法。使用本方法，计算得出酪氨酸酶的米氏常数（K）为1.347 mM。曲酸对游离酪氨酸酶的抑制常数（K）以及曲酸对酪氨酸酶/L-多巴复合物的抑制常数（K）分别计算为36.64和74.35 μM，并且确定曲酸的半数抑制浓度（IC）为46.64 μM。所开发的方法对于研究酶动力学、抑制动力学以及进一步筛选酶抑制剂而言快速且便捷。
