Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

Abstract

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.

Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants

“…This was shown using L-DOPA and dopachrome, the substrates of Tyr and Tyrp2, respectively. When incubated with dopachrome, which was produced in the lab using Tyr immobilized to the magnetic beads [21], two additional peaks appeared at 325 and 560 nm, which corresponded to the DHICA and IQCA, respectively; however, after the subtraction of the baseline, a significant level of DHICA only was detected (Figure 3B).…”

“…Here, Tyrp2 was incubated with L-DOPA or dopachrome for 1 h at 37 • C, and the absorption spectrum for the wavelength in the range of 200-900 nm was recorded (Figure 3A). Dopachrome was produced in the reaction of L-DOPA with Tyr immobilized to the magnetic beads [21]. In the presence of L-DOPA, the spectrum was on the baseline level; however, in the presence of dopachrome, two additional peaks appeared at 325 and 560 nm, which corresponded to DHICA and indole-5,6-quinone-2-carboxylic acid (IQCA), respectively.…”

Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutants of Tyrosinase

“…The WT, R422Q, and P406L proteins were purified as previously described [10,16]. Briefly, immobilized metal affinity chromatography (IMAC) was followed by two steps of size exclusion chromatography (SEC).…”

“…Recombinant truncated human wild-type tyrosinase, WT, and OCA1B-related mutant proteins, R422Q and P406L, were expressed and purified, as previously described [1,2,10,16]. Briefly, the WT (residues 19-469 of the full-length protein), R422Q, and P406L proteins were produced in whole insect Trichoplusia ni (T. ni) larvae.…”

“…Post-translational glycosylation ensures proper Tyr maturation, stability, and function. The active site of Tyr contains two copper atoms bridged by an aquo (hydroxo) ligand and ligated to six histidine residues (H180, H202, and H211 at CuA and H363, H367, and H390 at CuB) [3,9,10]. The stability of the copper ions within the active site of Tyr is reliant upon the presence of a bridging dioxygen molecule.…”