National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
NIH Shared Resources on Biomedical Engineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA.
Int J Mol Sci. 2021 Jan 13;22(2):734. doi: 10.3390/ijms22020734.
Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19-469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.
人酪氨酸酶（Tyr）是黑色素生成途径中的限速酶。Tyr 催化底物 L-DOPA 氧化成多巴色素和黑色素。目前，由于缺乏一种简单的方法将多巴色素与蛋白质部分分开，因此难以对多巴色素相关产物进行表征。在这里，我们将含有 6xHis 标签的催化纯重组人 Tyr 结构域（残基 19-469）固定在 Ni 负载的磁性珠（MB）上。透射电子显微镜显示 Tyr-MB 的直径在 168.2±24.4nm 范围内，而深棕色黑色素图像显示直径为 121.4±18.1nm 的单聚和多聚黑色素。使用 Hill 动力学，我们表明 Tyr-MB 具有与完整 Tyr 相似的催化活性。L-DOPA 的二酚氧化酶反应显示多巴色素形成的增加与 MB 的数量和温度有关。在 50°C 下，Tyr-MB 显示出一些残留的催化活性，表明固定化 Tyr 提高了蛋白质稳定性。相比之下，在 37°C 下，从 Tyr-MB 颗粒中分离出的多巴色素产物显示多巴色素呈橙棕色，与 L-DOPA、Tyr 和多巴色素混合物的颜色不同。将来，Tyr-MB 可用于大规模生产多巴色素和黑色素相关产品，并为眼皮肤白化病遗传疾病找到治疗方法。
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