| Melanins and amyloid-beta peptides are natural biological species produced in humans and other mammals; however their normal functions within their respective tissues are not fully understood, specifically with regard to metal chelation. Biophysical characterization of these naturally-occurring biological species forms the basis of the present work, and new insights into their roles within the context of Parkinson's and Alzheimer's diseases are presented and discussed.;First, calcium regulation in epidermal melanocytes is known to protect the cellular redox balance by regulating the supply of substrate, L-tyrosine, for melanogenesis. The pigment contained in these melanocytes, melanin, has been implicated in maintaining calcium homeostasis in the cell. Isothermal titration calorimetry (ITC) and fluorescence spectroscopy conducted on Sepia melanin herein support a role for melanin as an intracellular mediator of calcium homeostasis.;Second, neuromelanin (NM) isolated from the substantia nigra (SN) region of the human brain was studied by scanning probe and photoelectron emission microscopies. Atomic force microscopy reveals that NM granules are comprised of spherical structures with a diameter of ∼30 nm, similar to that observed for Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy data establishes a surface oxidation potential for NM similar to that reported for eumelanin, despite the presence of a significant fraction of the red pigment, pheomelanin, which is characterized by a higher oxidation potential. This data predicts a structural motif with pheomelanin at the core and eumelanin at the surface. Furthermore, NM isolated from other regions of the human brain (premotor cortex, cerebellum, putamen, corpus callosum, and globus pallidus) were studied by the same methods, and the results show that all NM's exhibit common morphological features and a consistent surface oxidation potential.;Third, copper ion binding to amyloid-beta (Abeta) peptides is implicated in the pathogenesis of Alzheimer's disease due to the accumulation of Cu 2+ in senile plaques, the presence of Abeta peptides as a major component of these plaques, and the ability of Abeta peptides to chelate and reduce Cu2+ ions in vivo. ITC is utilized to provide fundamental insight into the thermodynamics of Abeta-Cu 2+ complexes. Multiple sequence variants of the N-terminal, metal-ion-binding domain of Abeta, residues 1-16, are studied, focusing on the three His residues (H6, H13, H14), the N-terminus (D1), Tyr10, and the three mutations present in rat Abeta (R5G, Y10F, H13R). In contrast to humans, Abeta peptides naturally found in the brains of rats and mice do not normally form cerebral Abeta deposits with age. Because metal binding to human Abeta accelerates plaque formation, the differences in peptide sequence have been hypothesized to alter metal ion binding. We report that bidentate binding interactions at the N-terminus and H6, which form stable, six-membered chelate rings, are critical in the creation of a high-affinity binding site for Cu2+ ions. The neurobiological implications of these results are discussed herein.;Lastly, an accurate data analysis method for determining stoichiometry and thermodynamic parameters from ITC data for the binding of macromolecules to metal cations that are solubilized through an association with a weak ligand is presented. This approach is applied to determine the binding constant for the association of Cu2+ to the Alzheimer's Abeta peptide sequences studied herein. |
