Fig. 4: Time-course analysis of hTTR aggregation induced by subtilisin.

hTTR (1 mg/ml) was treated at 37 °C with subtilisin in TBS buffer, pH 7.4, containing 5 mM CaCl₂ at an enzyme:hTTR ratio of 1:20 (mol/mol). At time points, aliquots (50 µl) of the proteolysis reaction were withdrawn, diluted ten-fold with the same buffer and analysed by UV–Vis absorption spectroscopy and DLS. a UV–Vis absorption spectra of the proteolysis reaction of hTTR with subtilisin at increasing reaction time. Blank measurements with hTTR and subtilisin alone are also shown. b Plot of the Abs₂₆₀/Abs₂₈₀ ratio vs. time of a hTTR solution (0.1 mg/ml) in the presence (black circles) and absence (white circles) of subtilisin. The data points result from three different experiments. c DLS analysis of hTTR proteolysis by subtilisin after 24-h reaction. DLS traces of hTTR alone, after 0 or 24-h incubation are shown as controls. DLS data are reported as % intensity size distribution. For each trace, the values of R_h, %mass distribution and %PD of the oligomeric/polymeric species A, B, and C are reported in the Supplementary Table 1.