Horseradish peroxidase/H2O2 oligomerization of 5,6-dihydroxyindole to melanins:studied by ESI and MALDI mass spectrometry

Among natural pigments, melanins occupy an outstanding position because of their wide distribution throughout the phylogenetic scale, from lower invertebrates to mammals up to man, where they provide the major determinants of skin pigmentation. They are physiological metabolites present in the mammal brain as well as in the skin cells, being also involved in a variety of genetic and acquired pigmentary disorders, of great social concern. In the last decades, considerable progress has been made in all areas of melanin research, especially related to the molecular biology of pigment-related genes. A major remaining problem, however, is related to the pigments structure. Structural elucidations of melanins is of fundamental importance in understanding pigment cell metabolism: in fact, for example, changes in the biological function of these cells, might be responsible for malignant transformation as in melanoma genesis. The reason for such structural information lack is to be first ascribed to the problems associated with melanins isolation from natural sources, since they occur tightly bound to a protein matrix; also the unfavourable physicochemical properties of the pigments: i.e. poor solubility in particular make the conventional techniques for structural analysis and molecular weight determination very challenging. There is also increasing evidence that chemical heterogeneity, not only in the secondary but also in the primary structure of melanins, play a key role in defining the macroscopic properties of melanins. For these reasons, most of the information on the melanin structure rely upon in vitro studies of tyrosinase (or peroxidase)-catalyzed oxidation of melanin monomeric precursors. Mass spectrometry, which especially in the last years has been extensively used in the study of a large number of natural and synthetic polymers, has been poorly exploited in the case of melanins. In the last few years, the interest in mass spectrometry as a powerful analytical tool in melanin chemistry has significantly increased following the development of new ionization techniques among which MALDI-MS has been the most used in melanins characterization; on the other hand, no reports was present in the literature concerning the use of ESI mass spectrometry in this field. As to MALDI mass spectrometry, it has been exploited in the direct analysis of synthetic and natural occurring melanin oligomers and also in comparison of melanins deriving from different precursors. In this communication we will report on the study of the time course 5,6-Dihydroxindole oligomerization by horseradish peroxidise/H2O2 studied by MALDI-TOF and ESI mass spectrometry. By ESI-MS we have been able to detect oligomers up to trimers, after 1 minute of reaction, while by MALDI-MS the same reaction mixture revealed to be composed also by oligomers up to the heptamer. Finally we have been able to establish that the polymerization can go ahead, since after 1h of reaction oligomers up to the decamer have been detected. By increasing the reaction time also a series of peaks related to structurally degraded oligomers appears, according to previously reported data on the in vitro polymerization.