Aggregation studies of metallated and reduced SOD1 WT and SOD1 A4V on treatment with PB.

ThT fluorescence was monitored during the co-incubation of metallated SOD1 WT (50 μM) (a) or SOD1 A4V (50 μM) (b) with PB at 1:30 molar ratio at 37 °C with continuous shaking, under reduced conditions. The values were normalized to the maximal ThT intensity elicited by SOD1 WT or SOD1 A4V and fitted to a Boltzmann sigmoidal equation. The control and PB-treated species were compared to analyse the effects of PB on the aggregation of SOD1 WT and SOD1 A4V . Error bars indicate the standard deviation. Analytical SEC of metallated SOD1 WT (50 μM) (c) and SOD1 A4V (50 μM) (d) with PB were analyzed under reducing conditions before and after 24 hrs and 48 hrs incubation at 37ºC. The first peak eluting at the volume was denoted as oligomerized SOD1 WT /SOD1 A4V (L). The second, third, and fourth peaks were calculated as trimer (T), dimer (D), and monomer (M) species of SOD1 WT /SOD1 A4V running at 14.5 ml, 15.7 ml, and 16.5 ml, respectively. Size-exclusion chromatogram shows how PB binding to SOD1 WT /SOD1 A4V promotes shifting towards the formation of large aggregates (L) rather than toxic trimer (T) formation in comparison to the control (48hrs-TC, TC represents the Test Control). The monomer (M), dimer (D), trimer (T), and large aggregate (L) species are shown as green, purple, red, and black dotted lines, respectively. All the experiments were performed for n = 3 biological replicates.