Isolation and characterization of mammalian eumelanins from hair and irides

A new enzymatic procedure was developed for isolation of eumelanin from bla ck human hair which might provide a substantially intact pigment for struct ural characterization. Sequential digestion with protease, proteinase K and papaine in the presence of dithiothreitol afforded a pigment with a 6% w/w protein content. HPLC analysis of pyrrole acids resulting from alkaline H2 O2 degradation, carboxyl content determination, and ferricyanide titration showed that the isolated pigment is made up of 5,6-dihydroxyindole (DHI)- a nd 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units at a 6:1 rat io, exhibiting a significant degree of oxidative degradation. For compariso n, a different eumelanin isolated from black bovine irides by a similar enz ymatic procedure was analyzed. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the final pigment provided evidence for homolo gous series of DHICA oligomers, while chemical analysis allowed an estimate of 2:1 DHICA/DHI-derived units in the polymer, with a substantial proporti on of intact o-diphenolic functions. Iris melanin proved able to promote th e Fenton oxidation of deoxyribose while hair melanin was ineffective. Overa ll, these results provide, for the first time, unambiguous evidence for mar ked structural differences of mammalian eumelanins which may be directly re lated to the diversity of the sites of biosynthesis and storage, as well as to functional role of these pigments. (C) 2000 Elsevier Science B.V. All r ights reserved.