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

Abstract

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 the molecular composition of melanin. Herein, the absorption coefficients for melanosomes containing predominantly eumelanin, a polymeric pigment derived from the molecular precursors 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), are reported for λ = 244 nm. The absorption coefficient varies with the DHICA/DHI ratio, determined from chemical degradation analyses. With increasing DHICA content, the absorption coefficient of the melanosome 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 Eumelanin is a commonly occurring pigment derived from the molecular precursors 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA).1-4 Even though the molecular structure of eumelanin remains elusive, exquisite analytical techniques have been developed that enable determination of the relative contributions of these two precursors to the overall pigment. 2,5Studies of eumelanins from different tissues exhibit a wide range of DHICA/DHI ratios. [5][6][7][8] However, how such changes in the molecular composition affect the eumelanic UV absorption coefficient has not been examined for natural systems.

The UV‐Absorption Spectrum of Human Iridal Melanosomes: A New Perspective on the Relative Absorption of Eumelanin and Pheomelanin and its Consequences†

“…Wavelength‐dependent values for the absorption coefficients, εc , were determined as previously described (20,21,26). The total absorption of light, however, is a result of the absorption from all constituents within the melanosome: proteins, lipids and melanin.…”

“…An application of photoemission electron microscopy (PEEM) has enabled a direct measurement of the absorption properties of melanins within intact melanosomes (19–21). In this article, measurements of the absorption coefficients, εc , of intact iridal stroma melanosomes isolated from dark brown and blue–green human irides are described for the spectral range λ = 244–310 nm.…”

“…Quantitation of absorption coefficients. The method of using the PEEM image to calculate the absorption coefficient, εc , through the Beer–Lambert law ( ɛc = Aℓ −1 = log[ I 0 / I ] ℓ −1 ) has been previously published (20,21). In brief, values for the incident and transmitted intensities of light, I 0 and I , were determined from averages of the photoemission current of the portion of unobstructed substrate surrounding the shadow and the shadow region immediately behind the melanosomes, respectively.…”

“…In brief, values for the incident and transmitted intensities of light, I 0 and I , were determined from averages of the photoemission current of the portion of unobstructed substrate surrounding the shadow and the shadow region immediately behind the melanosomes, respectively. As previously discussed (20,21), it was the portion of the shadow that lies inside the first maximum of the diffraction pattern produced on the substrate that was used to calculate the average photoemission current of the shadow; it is this region that is void of significant scattering contributions. The value of the pathlength of the experiment was calculated as an average of the different choroid lengths through the melanosomes that corresponded to points in the observed shadows and was found through the following equation: …”

Light-Induced Stress as a Primary Evolutionary Driver of Eye Origins

“…Unless the enzyme Tyrp2 is present, only DHI is produced. Thus, it is likely that eumelanin first appeared as the lattice-like sheets of DHI, and Tyrp2 evolved later to help already stressed cells avoid the ROS associated with DHI synthesis by producing the more specialized and less taxing DHICA (Peles et al 2010;Galván and Solano 2015;Micillo et al 2017) . DHICA specifically absorbs light in the UV spectrum and acts as a potent ROS scavenger compared to the relatively ineffectual scavenger, DHI (Borovansky and Riley 2011) .…”