Figure 6.

Heparin induces rapid C-36 fibrillation and β-sheet aggregates with lower ThT fluorescence. (a) ThT traces of 8 μM C-36 in PBS show potent alteration of C-36 fibrillation by additions of heparin with lag phase and endpoint ThT reductions. Colored error bars indicate triplicate SD for each point. (b) A semilogarithmic plot of the of t_1/2 as a function of heparin_DS/peptide reveals a scaling relationship for ratios ranging from 0.02:1 to 1:1, correlating with the heparin-induced ThT fluorescence reduction. (c) A clear conversion to β-sheet is observed by CD (25 μM peptide) as a function of the heparin_DS/peptide. Inset shows the mean residue ellipticity change at 222 nm. (d) Time-resolved heparin-induced C-36 aggregation is followed by ThT and CD at 222 nm with 25 μM C-36 and heparin addition to ∼5:1 heparin_DS/peptide. Single exponential fits are shown for each curve with exclusion of the first three data points for the ThT signal (crosses). Inset illustrates increasing aggregation speed (normalized 222 nm CD signal) at higher heparin concentrations (29, 58, 115, 231, and 461 μM). The maximal extrapolated C-36 aggregation rate was 0.42 ± μM/s (Fig. S9e). (e) FITC-labeled heparin binds C-36 and is removed from solution. FITC-heparin concentration was 50 μg/mL, corresponding to 83 μM heparin_DS. The mean value for heparin_DS per C-36 peptide was 1.26 ± 0.04 (SE) based on the linear regression of two combined independent experiments. To see this figure in color, go online.