Direct chemical evidence for eumelanin pigment from the Jurassic period.

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.

Near-infrared excited state dynamics of melanins: the effects of iron content, photo-damage, chemical oxidation, and aggregate size.

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.

High-performance liquid chromatography estimation of cross-linking of dihydroxyindole moiety in eumelanin.

Eumelanin pigments consist of various ratios of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI). On alkaline hydrogen peroxide oxidation, these indole moieties give rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), respectively. In a recent study, we detected considerable amounts of other pyrrole acids, pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) and pyrrole-2,3,4-tricarboxylic acid (isoPTCA), among the oxidation products of fossil ink sacs more than 160 million years old. PTeCA and isoPTCA arise from the cross-linking of the DHI moiety of eumelanin at the C2 and/or C3 positions. We mimicked the process of cross-linking by heating synthetic eumelanins prepared from various ratios of DHICA and DHI at 100 °C for 18 days (or at 40 °C for 180 days). The heated eumelanins were analyzed after alkaline peroxide oxidation as PTCA, PDCA, PTeCA, and isoPTCA by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. On heating, PTCA decreased rapidly due to decarboxylation, whereas PDCA decreased gradually. Concurrently, PTeCA increased gradually to levels close to PTCA. IsoPTCA also increased gradually at lower levels. Similar changes were observed at 40°C at a much slower rate. These findings suggest that the PTeCA/PTCA ratio may serve as a good indicator of aging (cross-linking) of eumelanin.

Chemical characterization of pterosaur melanin challenges color inferences in extinct animals.

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.

Probing the surface calcium binding sites of melanosomes using molecular rulers.

Melanosomes have the capacity to bind significant concentrations of calcium, suggesting there are surface binding sites that enable cations to access the interior of fully pigmented melanosomes. The surface of melanosomes is known to contain significant concentrations of carboxylate groups which likely are the initial biding sites for calcium, but their arrangement on the surface of the melanosome is not known. In various calcium proteins, a bidentate coordination by two carboxylate groups is the most common structure. In this study, we determine the distance between neighboring surface carboxylic acid groups by examining the binding of a series of diamines (+)H3N(CH2)mNH3(+) (m = 1-5) to melanosomes isolated from the ink sacs of Sepia officinalis and bovine choroid tissue. Of these amines, ethylenediamine (m = 2) shows optimal bidentate binding, revealing a narrow distribution of distances between neighboring carboxylic acid groups, ∼480 pm, similar to that found in proteins for calcium binding motifs involving two carboxylate groups.

Pump-Probe Microscopic Imaging of Jurassic-Aged Eumelanin.

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.