Fibrillation of SOD1 is induced by mildly reducing conditions or small amounts of disulfide-reduced, apo SOD1 protein. (A) Increase in TfT florescence indicates fibrillation of 50 μM apo SOD1 in 10 mM potassium phosphate buffer, pH 7.4, incubated with constant agitation at 37°C in 96-well plates with 50 mM DTT (line 1, light gray), 1 M GdmHCl (line 2, medium gray), or no addition (line 3, black). (B) Electron micrograph of SOD1 fibrils generated in the presence of 5 mM DTT. (C) Disulfide-reduced SOD1 with free thiol groups initiates fibrillation in the absence of reducing agent. SOD1SH-SH and SOD1S-S generated during the lag phase of a fibrillation reaction in 5 mM DTT were purified by HPLC, either not treated or treated with N-ethylmaleimide (NEM) to block free thiols, and added to fibrillation reactions without reducing agent. Apo-SOD1 (47.5 μM) in phosphate buffer was incubated with 2.5 μM of SOD1 in the following forms: SOD12SH, ○; SOD1S-S, ▵; SOD12SH-4NEM, −; or SOD1S-S-2NEM, +. [Adapted and reprinted by permission of Chattopadhyay et al. (20)].