Different molecular constituents in pheomelanin are responsible for emission, transient absorption and oxygen photoconsumption

Steady-state absorption and emission spectroscopies, oxygen activation and transient spectroscopy on a single sample of synthetic pheomelanin are compared. The absorption, emission and excitation spectra of pheomelanin depend on the molecular weight (MW) of the dissolved pigment constituents. While weakly-depending on MW, the maximum of the emission excitation spectrum is approximately 400 nm. The electron paramagnetic resonance oximetry measurements show a clear increase in oxygen uptake between 338 and 323 nm, and also reveal a local enhancement around approximately 370 nm. Pump-probe absorption spectroscopy reveals that photoexcitation of pheomelanin by UVA light generates a transient absorption peak in the visible and UV regions within the instrument response. The action spectrum for the formation of the 780 nm transient species peaks at approximately 360 nm. While both transient absorption bands show the same ultrafast decay component, the 780 nm peak completely vanishes on the 10s of picosecond time scale, but the UV band shows a kinetic evolution to a subsequent intermediate. While in a similar wavelength range, the maximum of the action spectrum derived from the transient data, the emission excitation spectrum and the action spectrum for photoconsumption all differ from one another, suggesting that the chromophore responsible for each is not that same. This raises concern about comparing the results from different photochemical methodologies for melanin, which is a specific case of comparing data on systems where molecular constituents are not well defined.     

Photoionization thresholds of melanins obtained from free electron laser-photoelectron emission microscopy, femtosecond transient absorption spectroscopy and electron paramagnetic resonance measurements of oxygen photoconsumption

Free electron laser-photoelectron emission microscopy (FEL-PEEM), femtosecond absorption spectroscopy and electron paramagnetic resonance (EPR) measurements of oxygen photoconsumption were used to probe the threshold potential for ionization of eumelanosomes and pheomelanosomes isolated from human hair. FEL-PEEM data show that both pigments are characterized by an ionization threshold at 282 nm. However, pheomelanosomes exhibit a second ionization threshold at 326 nm, which is interpreted to be reflective of the benzothiazine structural motif present in pheomelanin and absent in eumelanin. The lower ionization threshold for pheomelanin is supported by femtosecond transient absorption spectroscopy. Unlike photolysis at 350 nm, following excitation of solubalized synthetic pheomelanin at 303 nm, the transient spectrum observed between 500 and 700 nm matches that for the solvated electron, indicating the photoionization threshold for the solubalized pigment is between 350 and 303 nm. For the same synthetic pheomelanin, EPR oximetry experiments reveal an increased rate of oxygen uptake between 338 nm and 323 nm, narrowing the threshold for photoionization to sit between these two wavelengths. These results on the solubalized synthetic pigment are consistent with the FEL-PEEM results on the human melanosomes. The lower ionization potential observed for pheomelanin could be an important part of the explanation for the greater incidence rate of UV-induced skin cancers in red-haired individuals.     

Probing near-infrared photorelaxation pathways in eumelanins and pheomelanins

Ultraviolet-visible spectroscopy readily discerns the two types of melanin pigments (eumelanin and pheomelanin), although fundamental details regarding the optical properties and pigment heterogeneity are more difficult to disentangle via analysis of the broad featureless absorption spectrum alone. We employed nonlinear transient absorption spectroscopy to study different melanin pigments at near-infrared wavelengths. Excited-state absorption, ground-state depletion, and stimulated emission signal contributions were distinguished for natural and synthetic eumelanins and pheomelanins. A starker contrast among the pigments is observed in the nonlinear excitation regime because they all exhibit distinct transient absorptive amplitudes, phase shifts, and nonexponential population dynamics spanning the femtosecond-nanosecond range. In this manner, different pigments within the pheomelanin subclass were distinguished in synthetic and human hair samples. These results highlight the potential of nonlinear spectroscopies to deliver an in situ analysis of natural melanins in tissue that are otherwise difficult to extract and purify.     

Spectroscopy and photoreactivity of trichochromes: molecular components of pheomelanins

The trichochromes are a class of small molecules present in pheomelanin (the red melanin) and absent in eumelanin (the black melanin). Herein trichochrome F (TF) and decarboxytrichochrome C (dTC) are examined. Both trichochromes are characterized by a visible absorption band, which is shown to be the result of overlapping transitions of the cis and trans isomers. The temperature dependence of the absorption spectrum of dTC suggests the additional presence of equilibrium between the enol and keto forms of the molecule. These conclusions are supported by ground-state energies of these isomers obtained using a continuum solvation model. Near-infrared emission measurements were not able to detect photoproduction of 1O2, and spin-trapping experiments revealed formation of O2*-. DNA nicking assays also revealed a low level of light-induced aerobic activity of dTC, suggesting a quantum efficiency of at most 5 x 10(-6) for the photogeneration of O2*-. These results are consistent with pump-probe optical experiments, which reveal efficient and nearly complete ground-state recovery within a few picoseconds of excitation. Both trichochromes are efficient quenchers of 1O2, exhibiting a bimolecular rate constant comparable with vitamin C. These results suggest that trichochromes could serve a protective role in pheomelanin pigments.     

Spectrophotometric methods for quantifying pigmentation in human hair-influence of MC1R genotype and environment.

Eumelanin (brown/black melanin) and pheomelanin (red/yellow melanin) in human hair can be quantified using chemical methods or approximated using spectrophotometric methods. Chemical methods consume greater resources, making them less attractive for epidemiological studies. This investigation sought to identify the spectrophotometric measures that best explain the light-dark continuum of hair color and the measure that is best able to distinguish red hair from nonred hair. Genetic analysis was performed on these two measures to determine the proportion of genetic and environmental influences on variation in these traits. Reflectance curves along the visible spectrum and subjective ratings of hair color were collected from 1730 adolescent twin individuals. Discriminant class analyses were performed to determine the spectrophotometric measure that could best proxy for eumelanin and pheomelanin quantities. The ratio of light reflected in the green portion of the spectrum to that reflected in the red portion of the spectrum was best able to distinguish red hair from nonred hair. Melanocortin 1 receptor (MC1R) genotype explained some, but not all, variation in this measure. Light absorbed in the red portion of the spectrum was best able to explain the light-dark continuum of hair color. Variance components analysis showed that there were qualitatively different genetic influences between males and females for the light-dark continuum of hair. Our results show that spectrophotometric measures approximating variation in eumelanin and pheomelanin may be considered as an alternative to chemical methods in larger epidemiological studies.     

Optical properties of human melanocytic nevi in vivo

We present an in vivo study of the optical properties of melanin present in melanocytic nevi of human subjects with Fitzpatrick skin type III (Caucasian descent) using optical spectroscopy. We show that the melanin absorption spectrum exhibits an exponential dependence on wavelength with a decay constant which follows a normal distribution characteristic of a random biological variable. Moreover, we demonstrate lack of correlation among melanin optical properties, melanin concentration and skin light scattering properties, which indicates that the true optical absorption of melanin can be measured free from confounding scattering effects. We also show that the average melanin absorption spectrum in vivo is in very good agreement with a previously reported oxygen photoconsumption action spectrum of melanin. Finally, we provide an overview of the emerging picture of the melanin absorption properties in vivo among various skin types and also among various skin lesions such as melanocytic nevi and melanoma.     

Investigating the Red Shift between in vitro and in vivo Urocanic Acid Photoisomerization Action Spectra

Abstract— Trans-urocanic acid (UCA) is found in the upper layer of the skin and UV irradiation induces its photoisomerization to cis -UCA. Cis -UCA mimics some of the immunosuppressive properties of UV exposure. The wavelength dependence for in vitro photoisomerization of trans-UCA (15 μM) over the spectral range 250 nm-340 nm (10 nm intervals) was determined. The action spectrum revealed that maximal cis-UCA production occurred at 280 nm, which is red-shifted by 10-12 nm from its absorption peak at 268 nm and differs markedly from the reported action spectra for cis-UCA production in mouse skin in vivo , which peaks at 300-310 nm. The reasons for the red shift between the in vitro and in vivo action spectra are not clear. There is limited evidence suggesting that the UV absorption maximum of trans- UCA red shifts from 268 nm in vitro to 310 nm on interaction with stratum corneum proteins in vivo. This phenomenon was investigated by applying trans-UCA (2.5 mg/cm²) in an oil emulsion to isolated human stratum corneum. After incubation at 37°C for 1 h, the absorption spectra of stratum corneum with UCA and with oil only were compared using a Xe arc source and a spectrora-diometer. A moderate red shift in trans-UCA absorption from ∼268 nm to 280 nm was observed. In summary, we suggest that the 10-12 nm red shift between the UCA absorption spectrum peak and the action spectrum peak in vitro may be accounted for by the wavelength dependence of quantum yields reported over the 254-313 nm range. The red shift between the in vitro and in vivo photoisomerization action spectra may result from the 10 to 12 nm red shift in the absorption of UCA in association with stratum corneum proteins, combined with increasing quantum yields over the 254-313 nm range.     

PHOTOINDUCED OXYGEN CONSUMPTION IN MELANIN SYSTEMS. ACTION SPECTRA AND QUANTUM YIELDS FOR EUMELANIN AND SYNTHETIC MELANIN

Abstract— The first quantitative measurements of the wavelength dependence of oxygen consumption in systems containing eumelanin (from bovine eyes) and synthetic DOPA melanin are reported. Consumption of oxygen (considered to be a requirement for immediate pigment darkening) during irradiation of melanins with either visible or ultraviolet light was monitored using a spin probe nitroxide-electron spin resonance spectroscopic approach. From initial rates of oxygen removal, quantum yields have been obtained over a wavelength range from 230 to 600 nm. Eumelanins are moderately effective in promoting oxygen consumption; quantum yields are low for irradiation with visible light, but increase sharply with light of shorter wavelengths. The absolute quantum yield for oxygen consumption is about 0.1% for natural melanin at 320 nm. The action spectrum is similar for both natural and synthetic melanins, indicating that the major chromophore responsible for oxygen consumption is the same for both kinds of material. This chromophore is not the major melanin chromophore responsible for absorption of visible light. The action spectrum also clearly differs from published action spectra for melanogenesis; however, the weak wavelength dependence for visible light is similar to that found for immediate pigment darkening. Catalase decreases the rate of oxygen consumption by 50%, confirming that hydrogen peroxide is the major molecular product of oxygen reduction. A Type I (free-radical) mechanism evidently predominates: D₂O and azide are shown to have only minor effects, ruling out any major contribution from a Type II (singlet-oxygen) process to the overall oxygen consumption.     

ISOLATION OF BLEPHARISMIN-BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step-up photophobic response) in response to light stimulation. A profile of a gel-permeation of a crude detergent-solubilized sample of the cells resulted in several red-colored fractions. Among these blepharismin-containing fractions, the fractions III-V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step-up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin-protein complex) is responsible for the step-up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore-protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin-layer chromatography-purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore-protein complex.     

Detection and quantitation of a pheomelanin component in melanin pigments using pyrolysis–gas chromatography/tandem mass spectrometry system with multiple reaction monitoring mode

Here, we describe the reliable method for the detection and quantitation of a pheomelanin component in melanin pigments. Synthetic melanins with various contents of pheomelanin‐type structural units were thermally degraded, and the multiple reaction monitoring mode was applied to detect the pheomelanin markers in the pyrolysates by GC/MS/MS. The method allowed the specific detection and quantitation of a pheomelanin component in melanin with the incorporation of pheomelanin‐type units as low as 0.05%. Considering highly universal character of the pheomelanin markers, the method could be applied for structural studies of natural melanin pigments being mixtures of eumelanin and pheomelanin. Copyright © 2012 John Wiley & Sons, Ltd.     

DUAL ROLE OF MELANINS AND MELANIN PRECURSORS AS PHOTOPROTECTIVE AND PHOTOTOXIC AGENTS: INHIBITION OF ULTRAVIOLET RADIATION-INDUCED LIPID PEROXIDATION

Ultraviolet radiation (UVR) is one of the risk factors for skin cancer and the main inducer of melanin pigmentation, the major protective mechanism of mammalian skin against radiation damage. The melanin pigments, eumelanin and pheomelanin, are likely to be important in protection against UVR, but their precursors are generally considered as phototoxic. The available data suggest DNA damage as the mechanism of phototoxicity. However, the effect of melanin precursors on membrane damage through lipid peroxidation, another important and probably more relevant (from the point-of-view of the melanosomal confinement of these molecules) mechanism of phototoxicity, is not known. As a model system for UVR–melanin–membrane interactions, we irradiated liposomes in the presence of eumelanin, pheomelanin and two of their major precursors, 5,6-dihydroxyindole (DHI) and 5-S-cysteinyldopa (SCD). The presence of the two melanin precursors substantially reduced the formation of lipid peroxidation products resulting from UVR exposure. The antioxidant activity of the melanin precursors was diminished under strong prooxidant conditions (presence of Fe³⁺). These results suggest that melanin precursors may have an important role in the protection of skin against the harmful effects of UVR including photocarcinogenesis.     

Gas chromatography–mass spectrometry analysis of pheomelanin degradation products

Melanoma is most rapidly increasing in the white population and people with pheomelanin skin type are at high risk to develop melanoma. However, little is known about the pheomelanin structure and function, and further elucidation of this melanin is therefore an important task. A GC/MS method was developed based on hydriodic acid hydrolysis of pheomelanin in the urine. Derivatization was performed with ethyl chloroformate and ethanol:pyridine (4:1, v/v). N,O-Ethoxycarbonyl-ethyl esters were extracted with chloroform and analyzed by GC/MS. 4-Amino-3-hydroxyphenylalanine and 3-amino-4-hydroxyphenylalanine together with one benzothiazinone and two benzothiazole compounds were detected and identified in hydrolyzed samples of synthetic pheomelanin and melanin from the urine of a patient with melanoma. These findings strongly suggest that heterocyclic pheomelanin-type units are incorporated in the pigment structures.     

Diffuse Reflectance Spectroscopy as a Reliable Means of Comparing Ultraviolet Radiation‐induced Erythema in Extreme Skin Colors

Erythema is widely considered an indicator of skin cancer susceptibility, but assessments are challenging in black skin because melanin can mask erythema under traditional visual and advanced objective Commission Internationale de l'Eclairage (CIE) L *a *b * assessments. Using spectral measurements (400–700 nm) from a spectrophotometer, an algorithm was developed to measure erythema in white Caucasians (n = 9) and black West Africans (n = 11) 19–24 h postsolar simulated radiation (SSR) exposures to the volar forearm. The derived spectrum achieved showed a strong maximum peak for hemoglobin at 580 nm and a linear slope between 650 and 700 nm for melanin absorption, as reported by other authors. Absorption by hemoglobin at 580 nm was used as a proxy for erythema, and melanin was quantified between 650 and 700 nm. Our algorithm corrected the erythema measurements for stray specular (mirror‐like) reflection and the melanin‐masking effect. A linear relationship between SSR exposure and erythema was evident (p < 0.0001 for white and black skin), and white skin is 8.4 times more responsive to SSR compared to black skin. The prediction of ultraviolet radiation sensitivity is vital in both clinical and investigative dermatology especially in the determination of starting phototherapy doses. Our methodology allows for the accurate assessment of erythema independent of constitutive pigmentation.     

PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

Abstract— A high resolution action spectrum for photoreactivation was determined using purified photoreactivating enzyme from Streptomyces griseus. Conversion of pyrimidine dimers in UV-irradiated DNA, the substrate for photoreactivating enzyme, was measured with a Haemophilus influenzae transformation assay. A high similarity was found between action spectrum (max. at 445 nm) and the long wavelength absorption band (max. at 443 nm)of photoreactivating enzyme. In addition to the400–470 nm region considerable photoreactivation was found with wavelengths between 280 and 320 nm. No evidence was obtained for the presence of nonenzymatic photoreactivation. Comparison of in vitro and in vivo action spectra revealed that the sharp peak at 313 nm found in vivo is probably the result of counteracting photoreactivation and inactivation effects. Comparison of the action spectrum with the absorption spectrum of 8-hydroxy-10-methyl-5-deazaisoalloxazine in an aprotic dipolar solvent (which serves as a model for the 8-hydroxy-5-deazaflavin chromophore in photoreactivating enzyme) indicates the possible presence of other chromophore(s) involved in the photorepair process. From kinetic measurements and flash experiments values were obtained for the rate constants of the photoreactivation reaction. The quantum yield of photoreactivation was estimated to be approximately 1.     

Melanin from epidermal human melanocytes: Study by pyrolytic GC/MS

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation.     

Probing the Motional Behavior of Eumelanin and Pheomelanin with Solid‐State NMR Spectroscopy: New Insights into the Pigment Properties

Melanin is the most widespread pigment in the animal kingdom. Despite its importance, its detailed structure and overall molecular architecture remain elusive. Both eumelanin (black) and pheomelanin (red) occur in the human body. These two melanin compounds show very different responses to UV‐radiation exposure, which could relate to their microscopic features. Herein, the structural properties and motional behavior of natural eu‐ and pheomelanin extracted from black and red human hair are investigated by means of solid‐state NMR spectroscopy. Several 1D and 2D NMR spectroscopic techniques were combined to highlight the differences between the two forms of the pigment. The quantitative analysis of the ¹H NMR wide‐line spectra extracted from 2D ¹H–¹³C LG‐WISE experiments revealed the presence of two dynamically distinguishable components in both forms. Remarkably, the more mobile fraction of the pigment showed a higher mobility with respect to the proteinaceous components that coexist in the melanosome, which is particularly evident for the red pigment. An explanation of the observed effects takes into account the different architecture of the proteinaceous matrix that constitutes the physical substrate onto which melanin polymerizes within the eu‐ and pheomelanosomes. Further insight into the molecular structure of the more mobile fraction of pheomelanin was also obtained by means of the analysis of 2D ¹H–¹³C INEPT experiments. Our view is that not only structural features inherent in the pure pigment, but also the role of the matrix structure in defining the overall melanin supramolecular arrangement and the resulting dynamic behavior of the two melanin compounds should be taken into account to explain their functions. The reported results could pave a new way toward the explanation of the molecular origin of the differences in the photoprotection activity displayed by black and red melanin pigments.     

Action spectra for photoinduced inactivation of bacteriophage T7 sensitized by 8-methoxypsoralen and angelicin

The action spectrum (240-300 nm) for photoinactivation of unsensitized phage T7 and the action spectra (310-380 nm) for photoinactivation of phage T7 sensitized with 8-methoxypsoralen (8-MOP) and angelicin were measured by an automated method. For unsensitized phage T7 the action spectrum is in good agreement with the absorption spectrum. For sensitization with angelicin the action spectrum is similar to the absorption spectrum, but for sensitization with 8-MOP the spectra are different. The agreement between the T7 absorption and action spectra in the far-UV region is due to photodamage of DNA, leading to phage inactivation. The similarity in the action and absorption spectra in the near-UV region for sensitization with angelicin seems to be in accordance with the monofunctional photobinding of angelicin to DNA. The action spectrum for sensitization with 8-MOP has a maximum at about 320 nm and this suggests that, in addition to the monoadducts, the biadducts play a role in the inactivation of phage T7. Taking the number of bound furocoumarin molecules into consideration, the quantum efficiencies were estimated. Furocoumarin increases the quantum efficiency in the near-UV region and the values are similar to those obtained in far-UV light without psoralens.     

The influence of melanins on the photoperoxidation of lipids

The influence of synthetic Dopa-melanin, natural-A, AB-melanin and pheomelanin has been studied for the following processes: (1) microsomal and liposomal lipid peroxidation induced by illumination with UV- and visible light; (2) interaction of melanin with the superoxide anion; and (3) chemiluminescence intensity of phospholipids in the presence and absence of melanins. It has been shown that (a) melanins can function as a scavenger of the superoxide anion radical O2-. (pseudo-superoxide dismutase) and (b) melanins protect microsomes and liposomes against lipid peroxidation which is due to the absorption of light energy (filter effect). Our data show that in the presence of melanins lipid peroxidation is significantly lower in comparison with the phospholipids incubated in the absence of melanins (except pheomelanin). The level of photoperoxidation of lipids was studied by means of the chemiluminescence method and by measurement of the malondialdehyde product. The results obtained by both methods indicate that melanin can effectively influence the process of lipid peroxidation. Mechanisms involved in the physical (optical screening) and photochemical reactions are proposed.     

Fast Redox Perturbation of Aqueous Solution by Photoexcitation of Pyranine

Abstract— Intense illumination (60-120 MW/cm²) of an oxygen-free aqueous solution of pyranine (8-hydroxypyrene-l,3,6-tri-sulfonate) by the third harmonic frequency of an Nd-Yag laser (355 nm) drives a two successive-photon oxidative process of the dye. The first photon excites the dye to its first electronic singlet state. The second photon interacts with the excited molecule, ejects an electron to the solution and deactivates the molecule to a ground state of the oxidized dye (φ⁺). The oxidized product, φ⁺, is an intensely colored compound (Λ_max= 445 nm, ε= 43 000 ± 1000 M ⁻¹ cm⁻¹) that reacts with a variety of electron donors like quinols, ascorbate and ferrous compounds. In the absence of added reductant, φ⁺ is stable, having a lifetime of -10 min. In acidic solutions the solvated electrons generated by the photochemical reaction react preferentially with H⁺. In alkaline solution the favored electron acceptor is the ground-state pyranine anion and a radical, φ, of the reduced dye is formed. The reduced product is well distinguished from the oxidized one, having its maximal absorption at 510 nm with e = 25 000 ± 2000 M^-l cm⁻¹. The oxidized radical can be reduced either by φ^- or by other electron donors. The apparent second-order rate constants of these reactions, which vary from 10⁶ up to 10⁹M⁻¹ s⁻¹, are slower than the rates of diffusion-controlled reactions. Thus the redox reactions are limited by an energy barrier for electron transfer within the encounter complex between the reactants.     

Probing the motional behavior of eumelanin and pheomelanin with solid-state NMR spectroscopy: new insights into the pigment properties

Melanin is the most widespread pigment in the animal kingdom. Despite its importance, its detailed structure and overall molecular architecture remain elusive. Both eumelanin (black) and pheomelanin (red) occur in the human body. These two melanin compounds show very different responses to UV-radiation exposure, which could relate to their microscopic features. Herein, the structural properties and motional behavior of natural eu- and pheomelanin extracted from black and red human hair are investigated by means of solid-state NMR spectroscopy. Several 1D and 2D NMR spectroscopic techniques were combined to highlight the differences between the two forms of the pigment. The quantitative analysis of the (1) H?NMR wide-line spectra extracted from 2D (1) H-(13) C LG-WISE experiments revealed the presence of two dynamically distinguishable components in both forms. Remarkably, the more mobile fraction of the pigment showed a higher mobility with respect to the proteinaceous components that coexist in the melanosome, which is particularly evident for the red pigment. An explanation of the observed effects takes into account the different architecture of the proteinaceous matrix that constitutes the physical substrate onto which melanin polymerizes within the eu- and pheomelanosomes. Further insight into the molecular structure of the more mobile fraction of pheomelanin was also obtained by means of the analysis of 2D (1) H-(13) C INEPT experiments. Our view is that not only structural features inherent in the pure pigment, but also the role of the matrix structure in defining the overall melanin supramolecular arrangement and the resulting dynamic behavior of the two melanin compounds should be taken into account to explain their functions. The reported results could pave a new way toward the explanation of the molecular origin of the differences in the photoprotection activity displayed by black and red melanin pigments.