Transition dipole strength of eumelanin

We report the transition dipole strength of eumelanin (the principal human photoprotective pigment) in the ultraviolet and visible. We have used both theoretical (density functional) and experimental methods to show that eumelanin is not an unusually strong absorber amongst organic chromophores. This is somewhat surprising given its role as a photoprotectant, and suggests that the dark coloring in vivo (and in vitro) of the eumelanin pigment is a concentration effect. Furthermore, by observing the polymerization of a principle precursor (5,6-dihydroxyindole-2-carboxylic acid) into the full pigment, we observe that eumelanin exhibits a small amount $(\sim 20\%)$ of hyperchromism (i.e., the reaction process enhances the light absorption ability of the resultant macromolecule relative to its monomeric precursor). These results have significant implications for our understanding of the photophysics of these important functional biomolecules. In particular, they appear to be consistent with the recently proposed chemical disorder secondary structure model of eumelanins.

Figure 1

The extinction coefficient of eumelanin vs frequency (solid) with multiple Gaussian fitting (dotted) (experimental spectrum as reported in Ref. 29). The chemical disorder model suggests that the broadband absorption spectrum of eumelanin can be reproduced by the summation of multiple Gaussian spectra with widths typical of solvent broadening for organic chromophores [9]. In order for this model to be consistent with the observed data, those species absorbing at higher energies must have either a higher abundance or larger oscillator strengths to produce the monotonic increase in the absorption towards higher energies.

Figure 2

The chemical structures of molecules relevant to this study. DHI and DHICA are the basic monomeric building blocks of the eumelanin macromolecule, whereas tyrosine is an earlier precursor. Fluorescein is a commonly used organic dye and quantum yield standard.

Figure 3

The extinction coefficients of synthetic eumelanin (solid), DHICA (dot-dash), tyrosine (dotted), and fluorescein (dashed). The inset shows DHICA, eumelanin, and tyrosine data with a different vertical scale (not visible on full plot due to the order of magnitude greater extinction coefficient of fluorescein compared to the other samples). For eumelanin, the extinction coefficient is expressed per monomer. Note the unusual broadband shape of the eumelanin spectrum as compared with the peaked spectra of the other organic molecules. These spectra are in qualitative and quantitative agreement with those in literature [8, 52, 53, 54]. The much greater magnitude of the fluorescein extinction coefficient is reflected in its much larger dipole strength (see Table ).

Figure 4

(Color online) Electron densities of important molecular orbitals as calculated by NRLMOL. The HOMO to $\mathrm{LUMO}+1$ transition is predicted to be that with substantial oscillator strength for tyrosine as shown in Table ; the tyrosine LUMO is also shown for comparison to DHICA and fluorescein. The HOMO to LUMO and $\mathrm{HOMO}-1$ to LUMO transitions are predicted to be those with substantial oscillator strengths for fluorescein and DHICA, as shown in Table . Red: oxygen, blue: nitrogen, green: carbon, white: hydrogen.

Figure 5

The extinction coefficient per monomer of DHICA as it undergoes oxidative polymerization to form eumelanin. The initially peaked DHICA spectrum gradually shifts towards the broadband eumelanin spectrum, but does not change substantially in integration area. Dipole strengths determined from this data are shown in Fig. 6.

Figure 6

The dipole strength per monomer of DHICA over time, as it reacts to form eumelanin, determined using different UV cutoff frequencies. The variation between the curves indicates that the absolute value of the dipole strength at each time point is only accurate to within an order of magnitude. The overall increase in dipole strength is, however, more robust, showing that eumelanin exhibits between 12 and 26 % hyperchromism (increase in dipole strength with polymerization).