Fig. 5: hTTR amyloid fibril formation during proteolysis with subtilisin.

hTTR (1 mg/ml) was treated at 37 °C with subtilisin in TBS buffer, pH 7.4, 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 At time points, aliquots (50 µl) of the proteolysis reaction were withdrawn, diluted ten-fold with the same buffer, and analysed by ThT binding assay and TEM. a Fluorescence emission spectra of ThT during proteolysis of hTTR (1 mg/ml) with subtilisin. At the indicated time points, aliquots of the proteolysis mixture were added with ThT (20 µM) and samples were excited at 450 nm and 25 ± 0.1 °C. b Time-course fluorescence intensity of ThT at 482 nm during subtilisin-induced proteolytis of hTTR (black circles). The data points result from three different experiments. The time-course emission of ThT, in the presence of intact hTTR alone (without subtilisin), is reported as a control (white circles). The fluorescence signal is expressed as F/F₀, where F₀ is the emission intensity of ThT at time = 0 min. c Representative TEM micrograph of hTTR fibrils, generated after 72-h incubation of hTTR (0.2 mg/ml) with subtilisin at 37 °C in 20 mM potassium phosphate, pH 7.6, 100 mM KCl. d For comparison, a representative micrograph of hTTR fibrils, generated after incubation of intact hTTR (0.2 mg/ml) under acidic conditions (0.2 M potassium acetate buffer, pH 4.4) for 72 h at 37 °C is also shown. The scale bar (100 nm) is indicated.