Figure 4

Slow oxidation of α-syn incubation in HEPES buffer only (a) and with Cu²⁺ (b). Incubation of 100 μM α-syn with or without Cu²⁺ only, showed the appearance of a dityrosine signal at 405 nm after 7 days of incubation which increases over 14 days. The presence of Cu²⁺ significantly increases dityrosine formation compared to buffer only. (c) ThT fluorescence assay to monitor fibril formation showed that the intensity increased significantly more for the α-syn incubated with 100 μM Cu²⁺ compared to α-syn without Cu²⁺ over 14 days. (d) TEM micrographs show comparison of α-syn incubated with and without Cu²⁺. The Cu²⁺ incubated α-syn assemblies display characteristic fibril morphology by negative stain TEM whilst sparse assemblies are observed for α-syn incubated without Cu²⁺. Immunogold labeling TEM for dityrosine cross-links reveals increased gold labels in α-syn incubated with Cu²⁺ and very few when Cu²⁺ is absent. Gold labels are evenly distributed along the fibrils (inserts).