• Sepia;
  • ethelenediaminetetraacetic acid;
  • magnesium;
  • calcium;
  • iron;
  • chelation;
  • aggregation


The structural organization of melanin granules isolated from ink sacs of Sepia officinalis was examined as a function of metal ion content by scanning electron microscopy and atomic force microscopy. Exposing Sepia melanin granules to ethelenediaminetetraacetic acid (EDTA) solution or to metal salt solutions changed the metal content in the melanin, but did not alter granular morphology. Thus ionic forces between the organic components and metal ions in melanin are not required to sustain the natural morphology once the granule is assembled. However, when aqueous suspensions of Sepia melanin granules of varying metal content are ultra-sonicated, EDTA-washed and Fe-saturated melanin samples lose material to the solution more readily than the corresponding Ca(II) and Mg(II)-loaded samples. The solubilized components are found to be 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-rich constituents. Associated with different metal ions, Na(I), Ca(II) and Mg(II) or Fe(III), these DHICA-rich entities form distinct two-dimensional aggregation structures when dried on the flat surface of mica. The data suggest multiply-charged ions play an important role in assisting or templating the assembly of the metal-free organic components to form the three-dimensional substructure distributed along the protein scaffold within the granule.