Ultraviolet Absorption Coefficients of Melanosomes Containing Eumelanin As Related to the Relative Content of DHI and DHICA

Central to understanding the photochemical function(s) of melanosomes is the determination of their absorption properties and an understanding of how the absorption varies with themolecular composition of melanin. Herein, the absorption coefficients for melanosomes containing predominantly eumelanin, a polymeric pigment derived from the molecular precursors 5,6-dihydroxyindole (DHI) and5,6-dihydroxyindole-2-carboxylic acid (DHICA), are reported forλ=244nm. The absorption coefficient varies with the DHICA/DHI ratio, determined from chemical degradation analyses.With increasing DHICA content, the absorption coefficient of themelanosome increases. This observation is consistent with reported extinction coefficients, which reveal that at 244 nm, the extinction coefficient of DHICA is a factor of ∼2.1 greater than that of DHI. The melanosome absorption coefficients are compared to predicted values based on a linear combination of the absorption of the constituent monomers. SECTION Biophysical Chemistry E umelanin is a commonly occurring pigment derived from the molecular precursors 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Even though the molecular structure of eumelanin remains elusive, exquisite analytical techniques have beendeveloped that enable determination of the relative contributions of these two precursors to the overall pigment. Studies of eumelanins from different tissues exhibit a wide range of DHICA/DHI ratios. However, how such changes in the molecular composition affect the eumelanic UV absorption coefficient has not been examined for natural systems. An understanding of these effects is critical as photoprotection is often invoked as one of the major biological functions of eumelanin. Approaches to date for determining the optical properties of intact melanosomes, the natural organelles containing eumelanin, involve indirect measurements and require assumptions about the thermodynamic properties of the intact organelle. For example, Jacques et al. utilized pulsed laser disruption to calculate internal absorption coefficients of several types of melanosomes. Using a pulsed laser to heat melanosomes andanalyzing themeasuredheatwith assumptions about melanosomal density and specific heat, they determined the wavelength-dependent melanosome absorption coefficient to be εc(λ) = 6.49 10(λ) cm. The lack of knowledge about the molecular structure of melanin precludes determination of c; therefore, only the product εc can be quantified currently. Thus, the melanosome absorption coefficient is reported as the product εc, which has units of inverse length. Photoemission electronmicroscopy (PEEM) canbe used to directly quantify the absorption coefficient of single, intact melansomes. Herein, we apply this method to measure the absorption cross sections of natural melanosomes comprisedpredominantlyof eumelaninbut of varyingDHICA/DHI ratios. This enables a quantitative examination of the relationship between the absorption coefficients of the intact natural melanosomes and their DHICA/DHI content. Figure 1 shows representative PEEM images. At the excitation wavelength, both the substrate and the melanosomes photoemit. Themelanosomes are brighter, however, because they have a greater photoionization cross section than the titanium substrate. The shape and dimensions of the observed melanosomes are consistent with previously reported scanning electron microscopy analyses. The substrate reveals shadows on the far side of the melanosomes along the direction of incident light (Figure 1a-f). The shadows reflect a decreased photoionization intensity of the substrate as a result of the absorption of the incident light by the melanosomes. An intensity contour plot of the shadow generated by the absorption of λ = 244 nm light by a bovine retinal pigment epithelial melanosome is shown in Figure 2. It is clearly observed that the shadow intensity of the image varies along the direction of the incident light and perpendicular to the Received Date: May 27, 2010 Accepted Date: July 13, 2010