Figure 5.

Characterization of the species formed during the aggregation reaction. 0.25 mg/mL bovine insulin was incubated at pH 2 in 50 mM NaCl and 60 °C, under quiescent conditions. To highlight the additional information gained with the methods described here, a “standard” kinetic trace is plotted monitoring the change in fluorescence intensity as a function of time when λ_ex = 360 nm and λ_em = 519 nm (a). Another dimension of information is added in (b), where the change in solvatochromic emission spectra is plotted as a function of time (indicated colorimetrically), with a constant excitation wavelength of 360 nm. The full three-dimensional spectra are plotted in Supporting Information Figure 5. An analogous aggregation reaction was monitored in real time as spectra were acquired, and aliquots were removed at time points (green dashed lines, (a)) whose spectra reveal the presence of aggregation intermediates. Normalized three-dimensional spectra for these time points are shown in (a). Representative AFM images are shown in (c). To generate the height/length plots (d), the features from generally between three and five images were identified and analyzed.