Analysis of apo-SOD1 aggregates by size-exclusion chromatography.

<p>Apo-SOD1 WT incubated in the absence (<b>A</b>) and presence of 250 μM of DHA (<b>B</b>), DHAOOH <b>(C)</b> or H₂O₂<b>(D)</b>. Apo-SOD1 G93A incubated in the absence <b>(E)</b> and presence of 250 μM of DHA <b>(F)</b>, DHAOOH <b>(G)</b>, or H₂O₂<b>(H)</b>. All experiments were conducted in the presence of 10 μM of the protein at 37°C for 24 h. The colored lines represent the incubation times: 2 h (black), 6 h (red) and 24 h (blue). Chromatograms are representative at least 3 independent experiments.</p

Nature of the apo-SOD1 aggregates formed in the presence of DHA or DHAOOH.

<p>Representative bis-ANS fluorescence spectra obtained for the control incubations containing DHA, DHAOOH or H₂O₂ without the protein (A); and for the incubations containing the (B) apo-SOD1 WT or (C) apo-SOD1 G93A incubated in the presence of DHA, DHAOOH or H₂O₂. Transmission electronic microscopy (TEM) of the apo-SOD1 WT or G93A mutant incubated with DHA (D). Two sets of images, one with the scale bar of 500 nm (upper panel) and the other of 100 nm (lower panel) are shown. Images are representative of two experiments. Representative visible spectra of congo red (CR) in the absence and presence of apo-SOD1 WT or G93A mutant pre-incubated with DHA or DHAOOH. Samples were added to the CR solution to give a final concentration of 6 μM of CR. All incubations were conducted in the presence of 10 μM protein and 250 μM of the lipid or H₂O₂ at 37°C for 24 h.</p

Role of Cys 6 and Cys 111 on DHA induced apo-SOD1 aggregation.

<p>C6S and C111S mutants of apo-SOD1 (10 μM) WT (<b>A</b>) and G93A (<b>B</b>) were incubated in the absence and presence of DHA (250 μM). Percentages of aggregates formed in the incubation were determined by size-exclusion chromatography analysis. The results were presented by means ± standard deviations of three experiments. Significant differences are indicated with * when <i>p<0</i>.<i>01</i>. Dots represent individual values.</p

Aggregation of apo-SOD1 WT and G93A in the presence of different fatty acids analyzed by size-exclusion chromatography.

<p>Typical chromatograms obtained for the incubations of apo-SOD1 WT (A) or G93A mutant (B) with different fatty acids. All incubations were performed with 10 μM of the protein and 250 μM of each fatty acid at 37°C for 24 h. Arrows indicate the SOD1 main peak and brackets indicate the SOD1 oligomers. Chromatograms are representative of at least 3 independent experiments.</p