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⁻¹ with LOD of 0.0851 U mL⁻¹. The proposal sensing system successfully afforded a prospective potential for application in enzyme kinetics and screening of inhibitor.

酪氨酸酶是酪氨酸代谢的关键酶，具有单酚酶活性和双酚酶活性。建立了一种实时荧光测定方法，通过硼酸盐和羟胺的组合消除双酚酶反应的干扰，专门监测单酚酶活性。根据质量守恒定律，由酪氨酸和多巴（L-3,4-二羟基苯丙氨酸）组成的合成基质与酪氨酸酶预孵育，总浓度为 70 μM，以模拟硼酸盐缓冲液中的单酚酶反应混合物。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，以消除来自多巴的光谱干扰。酪氨酸的检测限 (LOD) 为 0.61 μM。建立消耗酪氨酸的时间过程，通过实时读出反应混合物在比色皿中的酪氨酸荧光强度原位测量初始速度。该测定在 0.2839 至 1.7308 U mL 的单酚酶活性范围内起作用^-1 LOD 为 0.0851 U mL ^-1。该提议传感系统成功地提供了在酶动力学和抑制剂筛选方面的应用前景。