L bapna

Gingipains (RgpA, RgpB, Kgp) were purified from spent growth medium of P. gingivalis HG66 as described previously (47 (link), 48 (link)). Active-site titration with specific inhibitors KYT-1 (for RgpA/B) and KYT-36 (for Kgp) (Peptide Institute, Inc.) was performed using l-BApNa and N-(p-tosyl)-Gly-Pro-Lys-4-nitroanilide, respectively (Sigma-Aldrich), to determine the concertation of active gingipains (49 (link)). Substrate hydrolysis was measured at 405 nm for 40 min, and activity was presented as milli-optical density units per minute per microliter of sample. Before stimulation of eucaryotic cells, enzymes were activated with 10 mM l-cysteine as described recently (11 (link)).

Trypsin inhibitory activity and chymotrypsin inhibitory activity were measured essentially as previously described [50 ]. Briefly, chromogenic substrates (l-BAPNA, Na-benzoyl-l-arginine-p-nitroanilide from Sigma-Aldrich (B4875), and l-PAPNA, N-succinyl-l-phenylalanine-p-nitroanilide from Sigma-Aldrich (S4760)) were hydrolyzed by either trypsin or chymotrypsin in 50 mM Tris/HCl buffer (GE PlusOne Tris) of pH 8.5 in the presence of 5 mM CaCl₂ (Sigma-Aldrich C1016) in 96-well plates. This resulted in an increase in absorbance at 405 nm, which was monitored on a ThermoScientific Multiskan GO plate reader. Increasing quantities of inhibitor were added simultaneously to wells that were otherwise identical, in order to reduce the protease signal. The amount of protease that was rendered inactive per amount of inhibitor was then calculated by linear regression.

SVMPs, SVSPs and PLA2 enzymatic activities were assayed using synthetic substrates according to the procedures previously standardized in our lab [66 (link)]. For SVMPs, venom samples (10 $\mathsf{\mu }$ g) were incubated with 50 $\mathsf{\mu }$ M of Fluorescence Resonance Energy Transfer (FRET) Abz-AGLA-EDDnp substrate (GenOne Biotechnologies, Rio de Janeiro, RJ, Brazil), and the enzymatic reactions were monitored in a SpectraMax^® M2 fluorimeter (Molecular Devices, San Jose, CA, USA) with excitation at 340 nm and emission at 415 nm, at 37 ${}^{\circ }$ C in kinetic mode over 10 min with a read range of 1 min. The results were expressed in Relative Fluorescence Units-RFU/min/ $\mathsf{\mu }$ g. The PLA2 activity of venom samples (20 $\mathsf{\mu }$ g) was assayed using 500 $\mathsf{\mu }$ M of the substrate 4-nitro-3-[octanoyloxy] benzoic acid (Enzo Life Sciences, New York, NY, USA) incubated for 40 min at 37 ${}^{\circ }$ C and activity determined according to the absorbance at 425 nm and expressed as Absorbance/min/ $\mathsf{\mu }$ g of venom. For SVSPs, the venom was incubated using 800 $\mathsf{\mu }$ M of substrate N $\alpha$ -benzoyl-arginyl-p-nitroanide (L-BAPNA; Sigma-Aldrich, Darmstadt, Hesse, Germany) incubated for 40 min at 37 ${}^{\circ }$ C and according to absorbance at 405 nm and expressed as Absorbance/min/ $\mathsf{\mu }$ g. Differences in the enzymatic activity between both species were assessed using a One-Way ANOVA and considered statistically significant at p-value < 0.05.

In the case of serine proteinase, inhibition by plant extract was measured using benzoyl-arginyl-p-nitroanilide (L-BapNa) (Sigma-Aldrich) as a substrate of reference, as previously reported by Munekiyo and Mackessy [68 (link)] with some modifications. The work solution was prepared by dissolving 10.9 mg of BapNa in 250 µL of dimethylsulfoxide (DMSO–Sigma Aldrich) and 25 mL of buffer solution (10 mM Tris-HCl, 1 M CaCl₂, 10 mM NaCl, pH 7.8). The venom was dissolved in Tris-Hcl 50 Mm, pH 7.8 at a concentration of 1 mg/mL. To measure the activity, 20 μL of venom was mixed with 200 μL of substrate. Then, 25 μL of buffer A was added to the previous solution with 25 μL of extract in a 96-well microplate. These samples were incubated for 30 min at 37 °C. The plate reader was set at an absorbance of 405 nm. The absorbance was taken immediately after the incubation and 30 min later. The enzyme activity was determined by time differences in absorbance values.