A 160-kDa Protein Is Essential for Hemocyanin-derived Melanosis of Prawn

ABSTRACT A purified 160-kDa protein, isolated from the cuticle of kurama prawn and named melanosis collaborating factor (MCF), was a key factor in promoting melanosis by a cooperative reaction with hemocyanin-derived phenoloxidase but hemocyanin itself was incapable of producing black pigment. The enzymatic activity of MCF in the body of the prawn was very stable against the process of freezing and thawing; the enzyme maintained more than 80% of its activity after 3 mo of storage at −2 5 °C. These results indicated that MCF, acting in conjunction with hemocyanin-derived phenoloxidase, played a crucial role in postharvest melanogenesis in prawn.     

Age-dependent changes in eumelanin composition in hairs of various ethnic origins

Hair pigmentation is one of the most conspicuous phenotypes of humans. From a chemical point of view, however, data remain scarce regarding human hair pigmentation characteristics. To determine melanin content and composition in human eumelanic hair from individuals of different ethnic origins and at different ages, we collected hair from 56 subjects with eumelanic hair from each group of African-American, East Asian, and Caucasian origin. The 56 subjects consist of 14, seven each of males and females, each from four age classes of younger than 11, between 12 and 19, between 20 and 45, and older than 46. We analysed hair colour scale, total melanin value, and contents of pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA). We measured age-dependent increases in the relative quantity of eumelanin in pigmented human hairs in the three ethnic groups. Regarding melanin composition, we observed an increase in the PDCA/PTCA ratio with age in African-American and Caucasian hairs until approaching the quite constant level of the ratio in East Asian hairs in the elderly individuals. Our results evidence differences in the content and composition of eumelanin in human hair among African-American, Caucasian and East Asian individuals. Furthermore, we show evidence of age-dependent changes in the quantity and quality of eumelanin in pigmented human hairs. In particular, the age-dependent modification of the PDCA/PTCA ratio, a marker for 5,6-dihydroxyindole units in eumelanin, suggests a chronological evolution of hair follicle melanocyte phenotype (e.g. decrease in dopachrome tautomerase expression).     

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 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.     

“Fifty Shades” of Black and Red or How Carboxyl Groups Fine Tune Eumelanin and Pheomelanin Properties

Recent advances in the chemistry of melanins have begun to disclose a number of important structure-property-function relationships of crucial relevance to the biological role of human pigments, including skin (photo) protection and UV-susceptibility. Even slight variations in the monomer composition of black eumelanins and red pheomelanins have been shown to determine significant differences in light absorption, antioxidant, paramagnetic and redox behavior, particle morphology, surface properties, metal chelation and resistance to photo-oxidative wear-and-tear. These variations are primarily governed by the extent of decarboxylation at critical branching points of the eumelanin and pheomelanin pathways, namely the rearrangement of dopachrome to 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and the rearrangement of 5-S-cysteinyldopa o-quinoneimine to 1,4-benzothiazine (BTZ) and its 3-carboxylic acid (BTZCA). In eumelanins, the DHICA-to-DHI ratio markedly affects the overall antioxidant and paramagnetic properties of the resulting pigments. In particular, a higher content in DHICA decreases visible light absorption and paramagnetic response relative to DHI-based melanins, but markedly enhances antioxidant properties. In pheomelanins, likewise, BTZCA-related units, prevalently formed in the presence of zinc ions, appear to confer pronounced visible and ultraviolet A (UVA) absorption features, accounting for light-dependent reactive oxygen species (ROS) production, whereas non-carboxylated benzothiazine intermediates seem to be more effective in inducing ROS production by redox cycling mechanisms in the dark. The possible biological and functional significance of carboxyl retention in the eumelanin and pheomelanin pathways is discussed.     

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

Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene (TYR) is mutated in many cases of oculocutaneous albinism type 1 (OCA1). The mechanisms by which individual mutations contribute to the diverse pigmentation phenotype in patients with OCA1 have only began to be examined and remain to be delineated. Here, we analyze the temperature-dependent kinetics of wild-type Tyr (WT) and two OCA1B mutant variants (R422Q and P406L) using Michaelis–Menten and Van’t Hoff analyses. Recombinant truncated human Tyr proteins (residues 19–469) were produced in the whole insect Trichoplusia Ni larvae. Proteins were purified by a combination of affinity and size-exclusion chromatography. The temperature dependence of diphenol oxidase protein activities and kinetic parameters were measured by dopachrome absorption. Using the same experimental conditions, computational simulations were performed to assess the temperature-dependent association of L-DOPA and Tyr. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L followed by dopachrome formation is a complex reaction supported by enthalpy and entropy forces. We show that the WT has a higher turnover number as compared with both R422Q and P406L. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity and to discover novel therapies for patients.