Melanins are biological pigments found throughout the animal kingdom that have many diverse functions. Pump–probe imaging can differentiate the two kinds of melanins found in human skin, eumelanin ...and pheomelanin, the distributions of which are relevant to the diagnosis of melanoma. The long-term stability of the melanin pump–probe signal is central to using this technology to analyze melanin distributions in archived tissue samples to improve diagnostic procedures. This report shows that most of the pump–probe signal from eumelanin derived from a Jurassic cephalopod is essentially identical to that of eumelanin extracted from its modern counterpart, Sepia officinalis. However, additional classes of eumelanin signals found in the fossil reveal that the pump–probe signature is sensitive to iron content, which could be a valuable tool for pathologists who cannot otherwise know the microscopic distributions of iron in melanins.
Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions including display, evasion, photoprotection, ...detoxification, and metal scavenging. Two forms of melanin produced from different molecular precursors are present in nature—eumelanin (dark brown-black in color) and pheomelanin (orange-red in color). Both eumelanin and pheomelanin are complex highly cross-linked biopolymers that are found intertwined with proteins, lipids, and metal ions in nature. Recent reports have used morphological evidence to suggest the presence of melanin in the fossil record. These studies have been met with criticism due to their lack of chemical evidence to support melanin identification. This dissertation describes chemical approaches to unambiguously verify the presence of melanin in the fossil record and characterize the ancient pigment. It also explores the limitations for the survival of melanin in the fossil record and the possibility that melanin acts as a protective matrix to preserve other biomolecules that are embedded in the pigment. Melanin has unique chemical signatures that are commonly used to characterize and compare the pigment of modern organisms. We applied these chemical approaches to the study of fossil pigmentation. Analysis of the black pigmentation of two > 160 million year old (Mya) Jurassic cephalopod ink sacs provided the first conclusive evidence for eumelanin in the fossil record. The preserved fossil eumelanin was then compared to modern cephalopod eumelanin from Sepia officinalis. Using these chemical approaches we found that fossil eumelanin was chemically and morphologically identical to S. officinalis eumelanin. Although there is mounting chemical evidence for the presence and preservation of melanin in the fossil record, there is very little data constraining its long-term survival. We applied the analytical approaches designed to study fossil melanins and techniques used to study fossil sediments to compare the fossil inks from three deposits of similar age and lithology, but different maturation histories. Specifically, two ~ 180 Mya fossil ink sacs from a site that has entered the oil window in Holzmaden, Germany were compared to the previously analyzed fossil inks from two less mature sites in southern England. The chemistry of eumelanin was shown to alter at the onset of the oil window regardless of the age of the specimen. The decrease in surviving melanin was accompanied by an increase in the relative abundance of organic macromolecular material (kerogen), but no consistent change in melanin morphology. Finally, the role of melanin as a matrix that enhances the preservation of other biomolecules in the fossil record was considered. Proteins, commonly associated with melanin in modern organisms, were discovered in the aforementioned fossil ink sacs during full-scale chemical analysis. The amino acid profile of the protein in each fossil specimen was determined with an amino acid analyzer and compared to the amino acid profile the protein in modern S. officinalis. Statistical analysis of the amino acid distributions indicated that there is no significant difference between the amino acid profile of modern and fossil melanins. In order to verify the ancient origin of the amino acids in the fossil ink sacs, the ratio of D/L amino acid isomers was determined. While the proteins of living organisms consist of only L-amino acids, post-mortem the amino acids slowly convert from L to D form until they reach equilibrium (D/L = 1). This process is called racemization. The amino acids in the fossil ink sacs were racemized, which suggests their ancient origin. This marks the oldest determination of protein in a fossil system and provides evidence that the longevity of proteins may be enhanced when associated with melanin.
Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions, including display, evasion, photoprotection, ...detoxification, and metal scavenging. To date, evidence of melanin in fossil organisms has relied entirely on indirect morphological and chemical analyses. Here, we apply direct chemical techniques to categorically demonstrate the preservation of eumelanin in two > 160 Ma Jurassic cephalopod ink sacs and to confirm its chemical similarity to the ink of the modern cephalopod, Sepia officinalis . Identification and characterization of degradation-resistant melanin may provide insights into its diverse roles in ancient organisms.
•Comparison of fossil eumelanin reveals constraints on preservation.•Alteration of eumelanin is primarily due to maturation.•Alteration of eumelanin is largely independent of age and diagenetic ...mineralization.•Alteration of eumelanin is not accompanied by a change in granule morphology.
Melanins are polymeric phenolic pigments classified into two groups based on their chemical structures and molecular precursors: eumelanin (brown–black) and pheomelanin (yellow–red). Eumelanin is highly resilient and has a proven fossil record, extending back at least ∼200Ma. It is widespread in the biological world, occurring in fungi, the ink sacs of cephalopods, the feathers of birds, and the hair, skin, eyes, brain and inner ears of mammals. Although the presence and chemical attributes of fossil eumelanin have been documented, there are few data constraining its long term survival. Here we use a diversity of analytical techniques to compare the chemistry and morphology of fossilized cephalopod ink from three deposits of similar age and lithology, but different maturation histories. We demonstrate that the chemistry of eumelanin begins to alter at the onset of the oil window and is largely independent of age. The decrease in surviving melanin is accompanied by an increase in the relative abundance of organic macromolecular material (kerogen) but, critically for the correct interpretation of fossils, is not accompanied by a consistent change in granule morphology.
Melanosomes (melanin-bearing organelles) are common in the fossil record occurring as dense packs of globular microbodies. The organic component comprising the melanosome, melanin, is often ...preserved in fossils, allowing identification of the chemical nature of the constituent pigment. In present-day vertebrates, melanosome morphology correlates with their pigment content in selected melanin-containing structures, and this interdependency is employed in the color reconstruction of extinct animals. The lack of analyses integrating the morphology of fossil melanosomes with the chemical identification of pigments, however, makes these inferences tentative. Here, we chemically characterize the melanin content of the soft tissue headcrest of the pterosaur Tupandactylus imperator by alkaline hydrogen peroxide oxidation followed by high-performance liquid chromatography. Our results demonstrate the unequivocal presence of eumelanin in T. imperator headcrest. Scanning electron microscopy followed by statistical analyses, however, reveal that preserved melanosomes containing eumelanin are undistinguishable to pheomelanin-bearing organelles of extant vertebrates. Based on these new findings, straightforward color inferences based on melanosome morphology may not be valid for all fossil vertebrates, and color reconstructions based on ultrastructure alone should be regarded with caution.
Ultrafast pump–probe measurements can discriminate the two forms of melanin found in biological tissue (eumelanin and pheomelanin), which may be useful for diagnosing and grading melanoma. However, ...recent work has shown that bound iron content changes eumelanin’s pump–probe response, making it more similar to that of pheomelanin. Here we record the pump–probe response of these melanins at a wider range of wavelengths than previous work and show that with shorter pump wavelengths the response crosses over from being dominated by ground-state bleaching to being dominated by excited-state absorption. The crossover wavelength is different for each type of melanin. In our analysis, we found that the mechanism by which iron modifies eumelanin’s pump–probe response cannot be attributed to Raman resonances or differences in melanin aggregation and is more likely caused by iron acting to broaden the unit spectra of individual chromophores in the heterogeneous melanin aggregate. We analyze the dependence on optical intensity, finding that iron-loaded eumelanin undergoes irreversible changes to the pump–probe response after intense laser exposure. Simultaneously acquired fluorescence data suggest that the previously reported “activation” of eumelanin fluorescence may be caused in part by the dissociation of metal ions or the selective degradation of iron-containing melanin.
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