Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction
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 fro...
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2021-01-01
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| Series: | International Journal of Molecular Sciences |
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| author | Paul K. Varghese Mones Abu-Asab Emilios K. Dimitriadis Monika B. Dolinska George P. Morcos Yuri V. Sergeev |
| author_facet | Paul K. Varghese Mones Abu-Asab Emilios K. Dimitriadis Monika B. Dolinska George P. Morcos Yuri V. Sergeev |
| author_sort | Paul K. Varghese |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T04:57:01Z |
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| id | doaj.art-46851120496a425c92fb8f62b92c9468 |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T04:57:01Z |
| publishDate | 2021-01-01 |
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| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-46851120496a425c92fb8f62b92c94682023-12-03T13:04:44ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-01-0122273410.3390/ijms22020734Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic ReactionPaul K. Varghese0Mones Abu-Asab1Emilios K. Dimitriadis2Monika B. Dolinska3George P. Morcos4Yuri V. Sergeev5National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USANational Eye Institute, National Institutes of Health, Bethesda, MD 20892, USANIH Shared Resources on Biomedical Engineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USANational Eye Institute, National Institutes of Health, Bethesda, MD 20892, USANational Eye Institute, National Institutes of Health, Bethesda, MD 20892, USANational Eye Institute, National Institutes of Health, Bethesda, MD 20892, USAHuman 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.https://www.mdpi.com/1422-0067/22/2/734oculocutaneous albinismprotein purificationtyrosinasetyrosinase nanoparticlesdopachrome/melanin purification |
| spellingShingle | Paul K. Varghese Mones Abu-Asab Emilios K. Dimitriadis Monika B. Dolinska George P. Morcos Yuri V. Sergeev Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction International Journal of Molecular Sciences oculocutaneous albinism protein purification tyrosinase tyrosinase nanoparticles dopachrome/melanin purification |
| title | Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction |
| title_full | Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction |
| title_fullStr | Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction |
| title_full_unstemmed | Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction |
| title_short | Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction |
| title_sort | tyrosinase nanoparticles understanding the melanogenesis pathway by isolating the products of tyrosinase enzymatic reaction |
| topic | oculocutaneous albinism protein purification tyrosinase tyrosinase nanoparticles dopachrome/melanin purification |
| url | https://www.mdpi.com/1422-0067/22/2/734 |
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