Chemical Evaluation of Eumelanin Maturation by ToF-SIMS and Alkaline Peroxide Oxidation HPLC Analysis

Abstract

Residual melanins have been detected in multimillion-year-old animal body fossils; however, confident identification and characterization of these natural pigments remain challenging due to loss of chemical signatures during diagenesis. Here, we simulate this post-burial process through artificial maturation experiments using three synthetic and one natural eumelanin exposed to mild (100 °C/100 bar) and harsh (250 °C/200 bar) environmental conditions, followed by chemical analysis employing alkaline hydrogen peroxide oxidation (AHPO) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results show that AHPO is sensitive to changes in the melanin molecular structure already during mild heat and pressure treatment (resulting, e.g., in increased C-C cross-linking), whereas harsh maturation leads to extensive loss of eumelanin-specific chemical markers. In contrast, negative-ion ToF-SIMS spectra are considerably less affected by mild maturation conditions, and eumelanin-specific features remain even after harsh treatment. Detailed analysis of ToF-SIMS spectra acquired prior to experimental treatment revealed significant differences between the investigated eumelanins. However, systematic spectral changes upon maturation reduced these dissimilarities, indicating that intense heat and pressure treatment leads to the formation of a common, partially degraded, eumelanin molecular structure. Our findings elucidate the complementary nature of AHPO and ToF-SIMS during chemical characterization of eumelanin traces in fossilized organismal remains.

Sediment-encased pressure–temperature maturation experiments elucidate the impact of diagenesis on melanin-based fossil color and its paleobiological implications

“…These observations agree with previously published trends (Colleary et al 2015;Clements et al 2016) and suggest that loss of certain volatile/labile moieties concurrent with polymerization/cross-linking occurs during melanin diagenesis, especially with respect to reactive heteroatomic compounds such as N/S-bearing compounds. Similar cross-linking is also reported in maturation experiments on synthetically prepared eumelanin with differing dihydroxyindole and dihydroxyindole carboxylic acid units (Jarenmark et al 2021). We hypothesize that melanin undergoes thermal decomposition in several overlapping steps (i.e., volatile loss and polymerization co-occur, but different processes potentially dominate at different temperatures).…”

Preservation and Taphonomy of Fossil Insects from the Earliest Eocene of Denmark

“…Principal components analysis (PCA) of mass spectral data was conducted using the Solo software (version 7.9.5, Eigenvector Research, Inc., Manson, WA, USA), employing Poisson scaling and prior normalization of the peak intensities to the sum intensity of all included peaks. The analysis included all major "eumelanin" peaks (43 in total) in the mass range m/z 48-146 (see [49]). Reference spectra were acquired from pure calcium carbonate (Sigma-Aldrich Sweden AB, Stockholm, Sweden), eumelanin (from Sepia officinalis; Sigma-Aldrich Sweden AB) and synthetic eumelanin (Fisher Scientific GTF AB, Göteborg, Sweden).…”

“…mass range m/z 48-146 (see [49]). Reference spectra were acquired from pure calcium carbonate (Sigma-Aldrich Sweden AB, Stockholm, Sweden), eumelanin (from Sepia officinalis; Sigma-Aldrich Sweden AB) and synthetic eumelanin (Fisher Scientific GTF AB, Göteborg, Sweden).…”

Chemical and biochemical control of skin pigmentation with special emphasis on mixed melanogenesis

“…The biosynthetic methods have the strength of isolating various monomeric and oligomeric melanin intermediates, while the pulse radiolysis method is able to follow rapid reactions involving DQ. Some unsolved problems in the chemistry of mixed melanogenesis remain: (1) the nature of post‐polymerization modifications of EM and PM (such as photo and thermal modifications of EM and PM; Ito et al., 2018; Jarenmark et al., 2021); and (2) the nature of copolymerization of EM and PM. The biological functions of melanin are closely related to their structural features.…”