Analysis of melanins
Abstract: Mammalian melanins are of two major types, eumelanins and peheomelanins, which often occur together, then called mixed-type melanins. A method has been developed for analysing the various forms of melanins. Melanin characterisation included their oxidation in permanganate under alkaline conditions, and analysis of the resulting degradation products, pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and thiazole-4,5-dicarboxylic acid (TDCA). The PTCA/PDCA ratio manifested correlation with the relative proportions of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI) derived units in melanin, and the TDCA/PTCA+PDCA ratio good correlation with the relative proportions of pheomelanin and eumelanin in the pigment. The neuromelanin of the human substantia nigra was found to be of a mixed type, consisting of benzothiazines and indole units in about equal proportions. Melanin from cultures of IGR-1 melanoma cells was also of a mixed type with a eumelanin component ranging from 60 to 85 %. During oxidative stress, the cells manifested increased melanisation accompanied by a shift towards more indole residues with carboxygroups, as reflected by an increased PTCA/PDCA ratio. Fibroblasts transfected with the albino (c) locus gene and the brown (b) locus gene contained an enzyme with tyrosinase activity and another enzyme with dopachrome tautomerase activity. BOTH pheomelanin and eumelanin were synthesised in the transfected fibroblasts. An enzyme isolated from rat liver and human erythrocytes, was found to catalyse the formation of DHI from D-dopachrome. Another enzyme responsible for the tautomerisation of D-dopachrome to DHICA and isolated from the lens of bovine eye was identical with the immunoregolatory factor MIF. These enzymes were found useful in experiments making model melanins were the carboxycontents were predetermined.
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