Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, P. R. China.
Anal Chem. 2020 Apr 21;92(8):5780-5786. doi: 10.1021/acs.analchem.9b05127. Epub 2020 Apr 1.
Tyrosinase is the key enzyme in melanin biosynthesis and inherently involves both monophenolase activity and diphenolase activity. A continuous fluorometric assay method was developed for the first time to directly monitor the real monophenolase activity without the interference of diphenolase reactions through exclusively quenching the native fluorescence of DOPA by borate. Complexation with borate at pH 8.0 allowed for selective quantitation of tyrosine in a binary mixture of tyrosine and DOPA at 335 nm. The time course for consumption of tyrosine was established to measure the initial velocity by recording the tyrosine fluorescence intensity at discrete intervals. The assay worked in the monophenolase activity range from 0.13 to 2.01 U mL with the limit of detection (LOD) of 0.10 U mL. The assay method exhibited a promising prospect in application in kinetics of monophenolase and high throughput screening for monophenolase inhibitors.
酪氨酸酶是黑色素生物合成的关键酶，本质上同时具有单酚酶活性和二酚酶活性。本文首次开发了一种连续荧光检测法，通过硼酸特异性猝灭 DOPA 的天然荧光，可在不干扰二酚酶反应的情况下直接监测真实的单酚酶活性。在 pH8.0 时与硼酸络合，可在酪氨酸和 DOPA 的二元混合物中选择性定量酪氨酸，在 335nm 处测定。通过记录酪氨酸荧光强度在离散时间间隔的变化来建立酪氨酸消耗的时程曲线，以测量初始速度。该测定法可在 0.13 至 2.01 U mL 的单酚酶活性范围内工作，检测限（LOD）为 0.10 U mL。该测定方法在单酚酶动力学和单酚酶抑制剂高通量筛选方面具有广阔的应用前景。
