FIGURE 5.

Disulfide mapping of hedyotide B2. A, RP-HPLC separation of hedyotide B2 derivatives after partial S-reduction and S-NEM alkylation. Peak 1 is the native peptide; peaks 2 and 3 are intermediates with two intact disulfide bonds (2SS); peaks 4, 5, and 6 are intermediates with one intact disulfide bond (1SS); and peak 7 is the fully S-NEM alkylated hedyotide B2 (0SS). B, schematic presentation of hedyotide B2 unfolding pathway. Under our experimental condition, the Cys I–IV bond is likely to be reduced first, followed by the Cys II–V bond and lastly by Cys III–VI bond.