Porcine pancreatic trypsin

Porcine pancreatic trypsin (Sigma) and its substrate N_α‐Benzoyl–L‐arginine ethyl ester hydrochloride (Sigma) were used to measure the inhibitory activity of A1AT. Briefly, 300 ng of trypsin was incubated with or without 600 ng of A1AT in 40 µl of reaction buffer (15 mM Tris⋅HCl, pH 7.4, 100 mM NaCl, and 0.01% Triton X‐100) at 37°C for 30 min. After incubation, the solution was mixed with 960 µl of 0.25 mM α‐Benzoyl–L‐arginine ethyl ester hydrochloride in reaction buffer at room temperature. The absorbance of the reaction mixture at 405 nm was measured at 0 min and 5 min after reaction and the change in absorbance was used to calculate the remaining trypsin activity.

Rhesus rotavirus (RRV strain) and human astrovirus serotype 8 (HAstV-8 Yuc8 strain) were multiplied in MA104 or Caco2 cells, previously cultured as mentioned above. For this, 10 and 200 μg/ml porcine pancreatic trypsin (Sigma-Aldrich®/Cat# T-4799) were added to RRV and Yuc8 respectively, during 1 h at 37 °C in order to activate viral particles. After this time, soybean inhibitor (Life Technologies®/Cat# R-007-100) was added for 5 min at RT. Then, the activated viruses were added (adsorption period) to each cell line respectively, during 1 h in a 5 % CO₂ atmosphere at 37 °C. After one wash with PBS, fresh culture medium without FBS was added to complete 10 (RRV) and 12 (Yuc8) hour post-infection (hpi) respectively, until the immunodetection test (See below).
Anti-TLP (Triple – Layered Particles) polyclonal antibodies donated by Dr. Carlos Arturo Guerrero from Universidad Nacional de Colombia or anti-Yuc8 polyclonal antibodies donated by Dr. Ernesto Méndez^† from Instituto de Biotecnología – Universidad Nacional Autónoma de México were used to detect cytoplasmic viral antigens by immunocytochemistry or flow cytometry. The conjugates and substrates used were peroxidase – goat anti-rabbit IgG (Invitrogen™/Cat# 65-6120), AEC (3-Amino-9-ethylcarbazole – Sigma-Aldrich®/Cat# A5754) with hydrogen peroxide 0.02 %, and Alexa Fluor® 488 Goat (Life Technologies/Cat# A11034), respectively.

Standard multi-ionic solution for plasma spectrometry is from Perkin-Elmer, Waltham, MA, USA. N-benzoyl-DL-arginine, +catechin, p-nitroanilide, β-glucosidase, bacterial protease, porcine pancreatic trypsin, bovine pancreatic α-chymotrypsin, porcine intestinal peptidase, trypsin, boron trifluoride, and vainillin were from Sigma (Sigma Chemical Co., St Louis, MO, USA). All the other chemicals were of analytical grade.

IVPD was measured in triplicate using the pH-based Multi-Enzyme Technique suggested by Hsu et al. (11 (link)) with porcine pancreatic trypsin (Sigma T4799), bovine pancreatic chymotrypsin (Sigma C4129), and S. griseous protease type XIV (Sigma P5147), the latter as a substitute for porcine intestinal peptidase according to Hervera et al. (12 (link)). IVPD was calculated using the following equation:

Tinospora cordifolia stems were collected and their proteins were isolated. Enzymes were purchased from Sigma and Calbiochem, Merck. Pepsin was from porcine gastric mucosa (Calbiochem, Merck, activity: 3000 U/mg of protein calculated based on the substrate hemoglobin), Porcine pancreatic trypsin (Sigma, 10,000 U/mg of protein using the substrates benzoyl arginine ethyl ester, BAEE), bovine pancreatic α-chymotrypsin (Sigma, 40 U/mg of protein using the substrates benzoyl tyrosine ethyl ester, BTEE), porcine pancreatin (Sigma), and Soybean trypsin inhibitor (Calbiochem, Merck). The reagents required for the antioxidant assays were: 1,1-diphenyl-2-picrylhydrazyl(DPPH) (Sigma, minimum 95% purity by TLC), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (HPLC grade, Sigma), Ferrozine (extrapure, SRL) Ferrous chloride (extrapure, SRL), and Pyrogallol (HPLC grade, Sigma).