2.4. Surface plasmon resonance (SPR) spectroscopy

Kinetic analyses were performed using a Biacore T200 (GE Healthcare) at 25 °C. Amine coupling chemistry (NHS/EDC kit, GE Healthcare) was used to covalently immobilize SPINK1 and N34S mutant on a CM5 sensor chip surface (GE Healthcare). SPINK1 constructs were immobilized using a concentration of 10 μg/mL in 10 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer, pH 5.5 to a density below 40 response units (RU). Ethanolamine-inactivated flow cells were used for referencing and data was collected at 10 Hz. Human cationic trypsin (MW: 26.5 kDa; GenWay Biotech, San Diego, US) was prepared as two-fold dilutions in running buffer (100 mM Tris, 150 mM NaCl, 1 mM CaCl₂, 0.05% Tween20, pH 8.0 or 4.8, respectively). Trypsin was injected over the biosensor surface for 180 s followed by a dissociation time of 1000 s at a flow rate of 40 μL/min. The surfaces were regenerated after each cycle with regeneration solution (10 mM glycine/HCl, pH 2.0) for 100 s. Three independent experiments were carried out in each case. Data from six different concentrations between 1.56 and 50 nM were double referenced and fitted globally by a heterogeneous ligand model (BIAevaluation 3.1, GE Healthcare) to determine the binding parameters K_D1,2, k_on1,2 and k_off1,2.