Structural characterization of melanin from Black‐bone silky fowl (Gallus gallus domesticus Brisson)

Dear Sir, Melanin comprises black or brown pigments of high molecular weight formed by oxidative polymerization of phenolic or indolic compounds (Liu and Simon, 2003) and is widely distributed in the animal, plant and microorganism kingdoms. Natural melanin shows a wide range of biochemical activities, including photo-protection, antioxidation, free radical sequestration and immunomodulatory effects (Hung et al., 2002; Rozanowska et al., 1999; Sava et al., 2001). Animal melanin pigments can be classified into two major groups: the brown-toblack insoluble eumelanin and the yellow-to-reddishbrown pheomelanin, both of which derive from the common precursor dihydroxyphenylalanine o-quinone formed by tyrosinase oxidation of tyrosine (Nakagawa and Imokawa, 1996). Most animal melanin pigments present in pigmented tissues appear as mixtures or copolymers of eumelanin and pheomelanin (Wakamatsu and Ito, 2002). Extensive studies carried out over the years have led to the conclusion that eumelanin is highly heterogeneous polymer consisting of different oxidative states of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units, and the pyrrole units derived from their peroxidative cleavage, while pheomelanin consists of benzothiazine, benzothiazole and isoquinoline units arising from oxidative polymerization of cysteinyldopa isomers (Ito and Wakamatsu, 1998; Ito, 1986). The Black-bone silky fowl (BSF; Gallus gallus domesticus Brisson) is a special species of rooster originating in the Taihe County, east of Wushan Mountain in Jiangxi Province of China, and has been raised for over 2000 yr. Our previous study has shown that BSF contains high levels of carnosine, which is a powerful antioxidant and is taken as a dietary supplement in the West to strengthen muscles and to alleviate the effects of aging, autism and diabetes (Tian et al., 2007). A unique feature of the BSF when compared with the other common chickens (Muroya et al., 2000) is the presence of melanosomes in various organs including muscles, periosteum, trachea, mesentery, digestive canals, ovary and testis. However, the structure of melanin in the BSF has not been fully investigated. Here, we isolated and purified the melanin from the muscles of Taihe BSF by proteolytic extraction (papain, trypsin and pancreatin) without using harsh chemical reagents, and for the first time investigated the structure of BSF melanin using chemical degradation analysis and pyrolysis-gas chromatography ⁄ mass spectrometry (Py-GC ⁄ MS). Sepia melanin (without any treatment; Sigma Chemical Co.), a standard for natural eumelanin, was used as reference material. KMnO4 oxidation products of BSF melanin were analyzed with a high performance liquid chromatography–MS system. Pyrrole-2,3,5-tricarboxylic acid (PTCA), as a specific indicator of emelanin, was identified by monitoring the [M ) H] pseudo-molecular ion at massto-charge ratio of m ⁄ z 198. In the mass spectroscopy of KMnO4 oxidation products of BSF melanin, it was found that the collisional fragmentation of the PTCA precursor ion at mass-to-charge ratio (m ⁄ z) 198.03 produces abundant product ions at m ⁄ z 154.06 and 110.09 (Figure S1). These product fragments are formed by the loss of either one or two carboxylic acid functional groups from the PTCA molecule in the mass spectrometer collision cell. However, 1,3-thiazole-4,5-dicarboxylic acid derived from the benzothiazole units was not found in the KMnO4 oxidation products, which indicated that heterocyclic pheomelanin-type units are not incorporated into BSF melanin pigment structure. The yield of PTCA from BSF melanin by the permanganate oxidation method is smaller than Sepia melanin by a factor of 4.1, which indicates lower content of DHICA units in BSF melanin pigment compared to Sepia melanin. Similarly, the yield of PTCA by alkaline H2O2 oxidation is smaller than Sepia melanin by a factor of 4.2, and the yield of pyrrole-2,3-dicarboxylic acid (PDCA) by alkaline H2O2 oxidation is smaller than Sepia melanin by a factor of 2.87 (Table S1). As reported by Pezzella et al. (1997), Sepia pigment contains 75% of DHICA