Fig. 1: Proteolysis of hTTR by different proteases.

a hTTR (1 mg/ml) was reacted at 37 °C in TBS pH 7.4, containing 5 mM CaCl₂, with different proteases at an enzyme:hTTR ratio of 1:20 (mol/mol): trypsin and chymotrypsin, α-thrombin (αT), activated factors VII, IX, X, and XI, plasmin, human neutrophil elastase (HNE), cathepsin-G (Cat-G) and Proteinase-3 (Prot-3), subtilisin and neutral protease (NP) from Bacillus subtilis, thermolysin (TLN) from Bacillus thermoproteolyticus, and Glu-C specific endoproteinase (Glu-C) from Staphylococcus aureus. After 24-h reaction, the proteolysis mixtures were analysed by reducing SDS-PAGE (4–15% acrylamide) and Coomassie stained. hTTR was resistant to all proteases tested, except to subtilisin (red). Gels bands corresponding to the monomeric (M) and dimeric form (D) of hTTR are indicated. Shallow bands at molecular weights higher than hTTR dimers are assigned to the proteases added. b RP-HPLC analysis of the proteolysis reaction of hTTR with subtilisin. The reaction was conducted as in a. After 10-h reaction, the proteolysis mixture was acid-quenched and fractionated on an analytical C18 column. Fractions were collected and analysed by high-resolution MS, allowing to establish the chemical identity of the peptide material eluted in correspondence of the chromatographic peaks. Small fragments were identified in most cases. Of note, the fragment eluting with the major chromatographic peak was identified as Thr59-Glu127 peptide (7757.4 Da) and was found to be resistant to further proteolysis.