Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu, 610065, China.
Anal Bioanal Chem. 2021 Jan;413(2):635-647. doi: 10.1007/s00216-020-03034-4. Epub 2020 Nov 7.
Tyrosinase is the key enzyme for the metabolism of tyrosine and inherently comprises both monophenolase activity and diphenolase activity. A real-time fluorometric assay method was established to exclusively monitor the monophenolase activity by eliminating interference from diphenolase reactions through a combination of borate and hydroxylamine. Synthetic matrices comprised of tyrosine and DOPA (L-3,4-dihydroxyphenylalanine) preincubated with tyrosinase with the consistent sum concentration of 70 μM to mimic the monophenolase reaction mixture in borate buffer according to law of mass conservation. A matrix-matched calibration curve for determination of tyrosine was established using the synthetic matrices as standard sample to eliminate spectral interference from DOPA. The limit of detection (LOD) for tyrosine was 0.61 μM. The time course for consumption of tyrosine was established to measure the initial velocity through real-time reading out the tyrosine fluorescence intensity of the reaction mixture in a cuvette in situ. The assay worked in the monophenolase activity range from 0.2839 to 1.7308 U mL with LOD of 0.0851 U mL. The proposal sensing system successfully afforded a prospective potential for application in enzyme kinetics and screening of inhibitor. Graphical abstract.
酪氨酸酶是酪氨酸代谢的关键酶，本质上同时包含单酚酶活性和二酚酶活性。通过硼酸和羟胺的组合，建立了一种实时荧光检测法，通过消除二酚酶反应的干扰，专门监测单酚酶活性。合成基质由预先与酪氨酸酶孵育的酪氨酸和 DOPA（L-3,4-二羟基苯丙氨酸）组成，根据质量守恒定律，在硼酸缓冲液中模拟单酚酶反应混合物，一致的总浓度为 70μM。使用合成基质作为标准样品，建立了用于测定酪氨酸的基质匹配校准曲线，以消除 DOPA 的光谱干扰。酪氨酸的检测限（LOD）为 0.61μM。通过实时读取反应混合物在比色皿中的酪氨酸荧光强度，建立了酪氨酸消耗的时程，以测量初始速度。该测定方法在单酚酶活性范围为 0.2839 至 1.7308 U mL 之间，LOD 为 0.0851 U mL。该建议的传感系统成功地为酶动力学和抑制剂筛选的应用提供了潜在的前景。
