Bicarbonate enhances α-synuclein oligomerization and nitration:: intermediacy of carbonate radical anion and nitrogen dioxide radical

alpha-Synuclein, a neuronal presynaptic protein, has been reported to undergo oligomerization to form toxic Lewy bodies in neurodegenerative disorders. One of the proposed mechanisms for aggregation of alpha-synuclein involves oxidative and nitrative modifications. In the present study, we show that addition of 3-morpholino-sydnonimine chloride (SIN-1) or slow infusion of pre-formed peroxynitrite (ONOO-) to mixtures containing alpha-synuclein and HCO3- markedly enhanced both nitration and aggregation of alpha-synuclein through dityrosine formation. Bicarbonate-dependent peroxidase activity of Cu,Zn-superoxide dismutase (SOD1) also induced covalent aggregation of alpha-synuclein via a CO3.--dependent mechanism. Nitrone spin traps completely inhibited CO3.-- mediated oxidation/nitration and aggregation of alpha-synuclein. Conversely, alpha-synuclein inhibited CO3.--induced spin adduct formation. Independent evidence for CO3.--mediated oxidation and dimerization of alpha-synuclein was obtained from UV photolysis of [(NH3)(5)CoCO3](+), which generates authentic CO3.-. Irradiation of [(NH3)(5)CoCO3](+) and NO2- in the presence of alpha-synuclein yielded nitration and aggregation products that were similar to those obtained from a SIN-1 (or slowly infused ONOO-) and HCO3- or a myeloperoxidase/H2O2/NO2- system. Hydrophobic membranes greatly influenced alpha-synuclein aggregation and nitration in these systems. We conclude that both CO3.- and NO2. could play a major role in the nitration/aggregation of a-synuclein.