Figure 2

α-syn forms dityrosine cross-links in the presence of Cu²⁺/H₂O₂.

(a) 50 μM of monomeric α-syn was incubated for 24 h with Cu²⁺/H₂O₂ at 37 °C and agitation of 400 rpm. A fluorescence spectra was collected with excitation wavelength of 280 nm to explore tyrosine and dityrosine signals. Fluorescence spectra was also collected using excitation wavelength of 320 nm to focus on dityrosine signals and to record the early time points (inset). After one hour of incubation, the tyrosine fluorescence signal appeared to decline with simultaneous appearance of a new increasing signal at 405–410 nm, typical of the dityrosine fluorophore. (b) shows the development of dityrosine signal (at 405 nm) (ex. 320 nm) over time for 24 h and over 1 hour (inset). (c) Using an excitation wavelength of 280 nm, no change in the tyrosine signal and an absence of dityrosine fluorescence signal was observed over 24 h of incubation of 50 μM α-syn alone (i), with 50 μM Cu²⁺ only (ii) or with 1.25 mM H₂O₂ only (iii). (d) LC-ESIMS/MS (MRM) was used to detect dityrosine in the oxidized α-syn hydrolysate. The oxidized α-syn was obtained by oxidation of (50 μM) monomeric α-syn for 24 h with Cu²⁺/H₂O₂. Data is shown in a chromatogram as relative abundance against retention time showing the dityrosine standard for comparison.