Chemical characterization of eumelanins with special emphasis on 5,6-dihydroxyindole-2-carboxylic acid content and molecular size

Mammalian melanins exist in two chemically distinct forms: the brown to black eumelanins and the yellow to reddish pheomelanins. Eumelanins are derived from copolymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Eumelanins can be quantified by HPLC analysis of the oxidation product pyrrole-2,3,5-tricarboxylic acid (PTCA) and by our spectrophotometric method (Sp.EM) We also developed a spectrophotometric method for assaying the total amount of eu- and pheomelanins by dissolving them in Soluene-350 (TM). In addition, we previously showed that Sp.EM/TM and PCTA/TM ratios are significant parameters in characterizing eumelanins produced in follicular melanocytes. The objectives of this study were (1) to clarify the significance of Sp.EM/TM and PTCA/TM ratios in synthetic eumelanins and (2) to apply these methods to characterizing natural eumelanins with various DHI/DHICA ratios and molecular sizes. The results obtained show that (1) the Sp.EM/TM ratio of synthetic eumelanins increases as polymerization proceeds, (2) the Sp.EM/TM and PTCA/TM ratios in copolymers of DHI and DHICA correlate to the percentage content of DHICA-derived units, and (3) combination of the Sp.EM/TM and PTCA/TM ratios serves to estimate the DHICA content and the degree of polymerization in natural eumelanins. (C) 1997 Academic Press.