Bovine trypsin

The stoichiometry of inhibition against bovine trypsin (Sigma-Aldrich) was performe similarly as described^{31 (link),36 (link)}. Briefly, various concentrations of conserpin-AAT_RCL (0−200 nM in 25 nM increments) was incubated with a constant trypsin (105 nM) concentration at 37 °C for 30 min in 50 mM tris-HCl, 150 mM NaCl, 0.2% v/v PEG 8000 pH 8.0. The residual trypsin activity was measured at 405 nm using the substrate Na-benzoyl-L-arginine 4-nitroanilide hydrochloride (Sigma-Aldrich).
To test for activity after refolding, conserpin-AAT_RCL was unfolded in 6 M guanidine hydrochloride (GndHCl) 50 mM tris-HCl, 150 mM NaCl pH 8.0 for 2 hours before refolding via dilution for another 2 hours, so the final concentration of guanidine hydrochloride was 0.2 M. Any aggregate was pelleted by centrifugation and the sample dialysed against the same buffer to remove any remaining GndHCl. The SI assay against trypsin was performed as stated above (constant trypsin concentration of 210 nM and varying conserpin-AAT_RCL concentrations from 0−450 nM in 50 nM increments).
To observe an SDS-stable serpin: protease complex, different ratios of serpin were incubated with protease for 30 minutes at 37 °C. Reducing SDS sample buffer was added to each sample and quenched on ice to stop any further reaction. Samples were loaded onto a 10% SDS-PAGE.

Bovine trypsin (Sigma, T8642) was dissolved in 50 mM MES buffer (pH 6.0) containing 50 mM benzamidine and 1mM CaCl₂ to a final concentration of 20 mg/mL. 4 μL of protein solution was mixed with 4 μL of reservoir buffer (2.3 M (NH₄)₂SO₄ and 0.1 M MES pH 6.0) and equilibrated over the reservoir buffer at room temperature. Benzamidine-inhibited trypsin crystals were washed and equilibrated in an inhibitor exchange buffer (0.1 M MES, pH 6.0, 2.5 M (NH₄)₂SO₄ and 1 mM CaCl₂) for 6 hours to remove benzamidine. Washed crystals were then transferred to fresh inhibitor exchange buffer supplemented with saturating amounts of SFTI-TCTR and soaked for a further 48 hours. Crystals were washed 3 times in 10 μL of fresh inhibitor exchange buffer to remove surface bound inhibitor and transferred into the same buffer supplemented with 20% (v/v) glycerol for cryoprotection before being flash-cooled to 100 K in a nitrogen stream. Crystals were irradiated using a Cu Kα rotating anode source at 45 kV and 30 mA, and diffraction data was collected at 100 K from a Rigaku R-Axis IV⁺⁺ image plate. Indexing, scaling and merging of the data was performed by iMOSFLM [25 ] and Aimless [26 (link)]. All crystals were isomorphous with published SFTI-1/trypsin (PDB ID 1SFI) [15 (link)]. These coordinates were used as a starting structure for refinement with PHENIX [27 (link)] and Coot [28 (link)].

Gel sections were digested with bovine trypsin (Sigma) following reduction and alkylation of cysteine bonds with dithiothreitol and iodoacetamide (Sigma). Peptide extracts were subjected to chromatographic separation by C18 reversed-phase nano-trapping (Acclaim PepMap100 C18 Trap, 5 mm × 300 µm) and nano-analytical columns (EASY-Spray PepMap C18, 2 μm 100 Å, 75 µm × 15 cm) on an EASY NanoLC system (ThermoFisher) using a three-step linear gradient at a flowrate of 250 nl/min over 60 min. The eluate was ionized by electrospray ionization using an Orbitrap Velos Pro (ThermoFisher) operating under Xcalibur v2.2. Precursor ions were selected according to their intensity using the collision-induced fragmentation employing a Top20 CID method. Raw spectral data was processed using Proteome Discoverer (v1.4) against the Uniprot ‘All Taxonomy’ database under the Mascot 2.2 algorithm (Matrix Science). Results were analysed using Scaffold Software (version 4.11.0; Proteome Software).
This study was carried out in compliance with relevant institutional, national, and international guidelines, and legislation.

Formic acid (LC–MS grade), ammonium
hydrogen carbonate, bovine trypsin (sequencing grade), dithiothreitol
(DTT) (biochemistry grade), citrate dihydrate salt (biochemical grade),
and calcium dichloride were from Sigma Aldrich (St. Louis, United
States). Dichloromethane (DCM), acetonitrile (AcN) (HPLC and LC–MS
grade), methanol (MeOH) (HPLC synthesis or gradient grade), water
(LC–MS grade), and 2-propanol (2-PrOH) (LC–MS grade)
were from ChemLab (Bensheim, Germany). Trifluoroacetic acid (TFA),
Tris-(hydroxymethyl)-aminomethane (Tris), glycine, sodium chloride
(NaCl), acetic acid, and reduced or oxidized glutathione (GSH or GSSG,
respectively) were from Carl Roth (Karlsruhe, Germany). Endoprotease
ArgC was from Abnova (München, Germany).

To evaluate the effect of not fully digested proteins on DCs, purified γ-C [21 (link)] was hydrolyzed with bovine trypsin or with pepsin and pancreatin (Sigma-Aldrich, St. Louis, MO, USA) in an enzyme/protein ratio of 1:30, as previously described [29 (link)]. In brief, the γ-C protein was hydrolyzed with trypsin in ammonium bicarbonate (pH 7.5) for 45 min or with pepsin in 1 mM HCl (pH 3.0) for 45 min followed by hydrolysis with pancreatin in ammonium bicarbonate (pH 7.5) for 45 min. The hydrolysis was stopped by heating for 30 min at 65 °C, and samples were freeze-dried for subsequent use.
The purity of the γ-C protein was assessed through SDS-PAGE analysis (Figure S1). γ-C digested samples were analyzed by RP-HPLC using a SIMMETRY300 C18 (5 µm) (4.6 mm × 250 mm) column (Waters, Sesto San Giovanni, Italy) fitted on a chromatographic apparatus (Waters) composed of two 510 HPLC Pumps, a 717plus Autosampler, and a 996 Photodiode Array Detector. The mobile phase flux was 0.8 mL/min, mixing solutions A (TFA 0.1% in water) and B (TFA 0.1% in ACN) as follows: 2 min isocratic 100% solution A, 50 min linear gradient to 25% solution A, and 75% solution B. Peaks were detected at 220 and 280 nm.

A possible inhibitor or enhancer of trypsin/chymotrypsin activity present in
venom fraction was tested. The reaction mixture was prepared as described by the
supplier (PBS, enzyme, venom and chromogenic substrate specific for each
enzyme). The assay was performed with bovine trypsin and α-chymotrypsin (1 mg/50
mL, 0.001 M HCl; Sigma-Aldrich Co., USA), trypsin and α-chymotrypsin substrates
(10 mg/mL N-p-Tosyl-Gly-Pro-Lys-4-nitroanilide acetate salt and
0.2 mg/mL N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide,
respectively; Sigma-Aldrich Co., USA), phosphate-buffered saline (PBS) pH 7.4,
and Fraction I at two concentrations (1.90 mg/mL and 5.65 mg/mL). The substrate
solution was prepared at the 1 mg/20 mL assay concentration in PBS. Volumes of 5
μL of bovine trypsin and α-chymotrypsin were tested with 5 μL of substrate in
the 96-well samples at room temperature with 100 μL PBS. The reaction mixtures
were read at 410 nm to quantify the formation of p-nitroaniline (yellowish
color) every 2 minutes during 200 minutes.