FIG. 4.

Size-exclusion chromatography of reduced and oxidized peptides. Peptides 80-94 and 83A were analyzed by size-exclusion chromatography with a prepacked Superdex Peptide HR 10/30 column (Amersham Biosciences). A total of 50 μl of peptide solution (0.5 mg/ml) was injected onto the column and eluted with PBS. Solid lines, peptide solutions with minimal oxidation times (diluted in PBS and frozen for <15 min after they were dissolved in oxidation buffer); dashed lines, peptides oxidized to ≥95% (<95% of the original free sulfhydryl content by the Ellman assay). Oxidized samples were also diluted in 50 mM DTT to reduce any disulfide bonds and were then applied to the column (dotted lines). Although some sample-to-sample variability in the amounts of peptides eluting at each point was seen, substantial dimer peaks were observed only in oxidized samples, and aggregates eluting at 9.0 ml were seen only in fresh, nonoxidized samples. mAU, milli-absorbance units