Reovirus (~2.2 × 109 particles per reaction) was diluted in VDB containing human trypsin (BioVision, P1228-1000), bovine trypsin (Sigma, T-8003), porcine trypsin (Sigma, T4799), human chymotrypsin (Sigma, SRP6509), or bovine chymotrypsin (Sigma, C3142) at a final concentration of 14 µg/mL. Alternatively, the same number of reovirus particles was added to cathepsin reaction buffer (50 mM sodium acetate pH 5.0, 3 mM DTT, 100 mM NaCl) containing either 4 µM of mouse cathepsin B or 1 µM of mouse cathepsin L, as previously described (35 (link)). Reaction mixtures were incubated at 37°C for the indicated timepoints before being processed for Western blot analysis after the addition of 5× protein sample buffer and boiling.
Bovine chymotrypsin
Manufactured by Merck Group
Sourced in United States, Canada, Belgium
Bovine chymotrypsin is a lab equipment product that functions as a proteolytic enzyme. It is derived from the pancreas of bovine (cattle) and is commonly used in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Bovine trypsin, by Merck Group (3 mentions)
Porcine pepsin, by Merck Group (2 mentions)
Porcine trypsin, by Merck Group (2 mentions)
Synergy htx multi mode microplate reader, by Agilent Technologies (1 mentions)
N methoxysuccinyl ala ala pro val p nitroanilide, by Merck Group (1 mentions)
Porcine elastase, by Merck Group (1 mentions)
Human cathepsin l, by Merck Group (1 mentions)
Polarstar omega spectrophotometer, by BMG Labtech (1 mentions)
Papain, by Merck Group (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Azo cm cellulose azo cmc, by Megazyme (1 mentions)
Xylan, by Merck Group (1 mentions)
Curdlan, by Megazyme (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Laminarin, by Merck Group (1 mentions)
Pnp β d glucopyranoside, by Merck Group (1 mentions)
Xyloglucan, by Megazyme (1 mentions)
Trypsin, by Merck Group (1 mentions)
7 protocols using bovine chymotrypsin
1
Reovirus Particle Proteolytic Processing
2
Cyanobacteria Extract Inhibition Assay
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Cyanobacteria extract (1 mg/ml, methanol) was diluted in 10 mM ammonium acetate pH 4.5 to 30 ug/mL and then sequentially diluted 1.5-fold to 0.52 µg/mL in the same buffer. Bovine chymotrypsin (Sigma Aldrich) and Suc-Ala-Ala-Pro-Phe-AMC (Calbiochem, 230914) were diluted to 300 nM and 150 µM, respectively in 10 mM ammonium acetate pH 4.5. In a 384-well black microplate, 10 µL of enzyme, substrate, and cyanobacteria extract were combined (30 µL final volume) such that the concentrations in the reaction were 100 nM chymotrypsin, 50 µM of Suc-Ala-Ala-Pro-Phe-AMC and 10 µM to 0.17 nM of cyanobacteria extract. For solvent compatibility assays, chymotrypsin (100 nM) was assayed with 10 µg/mL and 1 µg/mL extract in 10 mM ammonium acetate, pH 4.5 containing 8.3 to 41.7% acetonitrile. A control assay lacked acetonitrile and cyanobacteria extract. All assays were performed in triplicate wells at 25 °C in a Synergy HTX Multi-Mode Microplate Reader (BioTek, Winooski, VT) with excitation and emission wavelengths of 360 and 460 nm, respectively. The initial reaction velocity in each well was recorded and the dose-response curve was generated using GraphPad Prism 9 software.
3
Amidolytic Activities of Venom Proteins
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The amidolytic activity was determined using 50 μg of venom protein, to measure trypsin-like, chymotrypsin-like, and elastase-like activities using BApNA, SAAPFpNA, and 10 mM N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) in dimethylsulfoxide as substrates, respectively. The mixture was brought to a final volume of 120 μL with 0.1 M Tris (pH 8) buffer and 10 μL of substrate was added to start the reaction. Activity was determined by the release of p-nitroanilide that absorbs at 405 nm, reporting as activity units (AU)/min/µg venom protein. One AU corresponded to an increase of absorbance of 0.01 at 405 nm [68 (link)]. Analyses were done in triplicate. In each case, bovine chymotrypsin, bovine trypsin and porcine elastase (Sigma-Aldrich) were used as positive controls.
4
Inhibition Kinetics of Cathepsin Proteases
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Enzymes included bovine trypsin, bovine chymotrypsin, human cathepsin B, human cathepsin L, and human cathepsin S (all Sigma-Aldrich) and human cathepsin K (Enzo Life Sciences). Purified F. hepatica cathepsin L1 (FhCL1), F. hepatica cathepsin L2 (FhCL2) and F. hepatica cathepsin L3 (FhCL3)66 (link) and recombinant FhKT1 and FhKT1Leu15/Arg1510 (link) were expressed as active recombinant proteins in P. pastoris. Reaction conditions and substrates employed for measuring the activity of each protease were as reported by Smith et al.10 (link). Additionally, rFhCL3 activity was measured using the fluorogenic substrate Z-Gly-Pro-Arg-NHMec (20 μM).
KT inhibitors (2 μM) was incubated with each protease in a 100 μl volume of reaction buffer for 15 min at 37 °C. Reaction were brought to 200 μl with the addition of fluorogenic substrate dissolved in reaction buffer and proteolytic activity measured as RFU (relative fluorescent units) using a PolarStar Omega spectrophotometer (BMG LabTech, UK). Inhibition constants were determined using the Morrison equation for tight-binding inhibition as previously described10 (link).
KT inhibitors (2 μM) was incubated with each protease in a 100 μl volume of reaction buffer for 15 min at 37 °C. Reaction were brought to 200 μl with the addition of fluorogenic substrate dissolved in reaction buffer and proteolytic activity measured as RFU (relative fluorescent units) using a PolarStar Omega spectrophotometer (BMG LabTech, UK). Inhibition constants were determined using the Morrison equation for tight-binding inhibition as previously described10 (link).
5
Purification and Characterization of Proteases
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Papain (2 × crystallized) (EC 3.4.22.2), ficain (EC 3.4.22.3), bovine rennin (3.4.23.4), porcine pepsin (3.4.23.1), rhizopuspepsin, bovine chymotrypsin (EC 3.4.21.1), bovine trypsin (EC 3.4.21.4), and porcine kallikrein (EC 3.4.21.35) were obtained from Sigma-Aldrich (St. Loius, MO, USA), bovine thrombin (EC 3.4.21.5) from Calbiochem (La Jolla, CA, USA), Bacillus subtilis subtilisin (EC 3.4.21.62) from Boehringer Mannheim (Mannheim, Germany) and porcine elastase (EC 3.4.21.36) from Serva (Heidelberg, Germany). Legumain (EC 3.4.22.34) was isolated from germinated bean seeds as previously described [33 ] and recombinant human cathepsin L (EC 3.4.22.43) and cathepsin H (EC 3.4.22.16) were prepared as described [9 (link)]. Haemonchus contortus proteases APR1 and PEP1 were prepared recombinantly in insect cells [34 ]. Substrates benzyloxycarbonyl(Z)-Phe-Arg-MCA (7(4-methyl)-coumarylamide), Arg-MCA, Z-Ala-Ala-Asn-MCA, Suc-Ala-Ala-Pro-Phe-MCA, Boc-Gly-Arg-Arg-MCA, H-Pro-Phe-Arg-MCA, t-butoxycarbonyl-Val-Pro-Arg-MCA, and Suc-Ala-Ala-Ala-MCA were from Bachem (Bubendorf, Switzerland), and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys-(Dnp)-NH2 was from Peptide Institute Inc. (Osaka, Japan). Synthetic cysteine protease inhibitor E-64 and the aspartic protease inhibitor pepstatin A were from Peptide Institute Inc.
6
Cellulose Preparation and Characterization
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QIAquick PCR purification kit and QIAamp DNA stool mini kit were all purchased from QIAGEN (Toronto, ON, Canada). AZO-CM-cellulose (AZO-CMC), curdlan, xyloglucan and GH5 family cellulase from Bacillus amyloliquefaciens (BamGH5A) were from Megazyme (Wicklow, Ireland). Carboxymethyl cellulose (CMC, low viscosity), hydroethyl cellulose (HEC), Avicel (PH101), laminarin, xylan from birch wood, locust bean gum (LBG), pNP (p-nitrophenol)-β-D-cellobioside, pNP-β-D-glucopyranoside, IPTG, porcine trypsin and bovine chymotrypsin were all purchased from Sigma-Aldrich (Oakville, ON, Canada). pCR4Blunt-TOPO vector kit and Pfx Ultra DNA polymerase were from Invitrogen (Burlington, ON, Canada). Restriction enzymes and T4 DNA polymerase and ligase were from Fermentas (Burlington, ON, Canada) . All other chemicals were of analytical grade and were obtained from either Sigma-Aldrich or Fisher Scientific (Nepean, ON, Canada). The regenerated amorphous cellulose (RAC) was prepared from the avicel (PH101) as previously described by Zhang et al.61 (link).
7
Simulated Digestion of Chickpeas and Lentils
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Dried raw Kabuli-type chickpeas (C. arietinum) harvested in Argentina in 2018, were donated by Greenyard Prepared (Bree, Belgium). Dried raw Dupuy-type green lentils (L. culinaris) grown and harvested in Canada in August 2019, were donated by Casibeans (Melsele, Belgium). Both materials (<10% moisture) were sorted to remove foreign material, and seeds were stored at -40°C, below their glass transition temperature, ensuring stability (Kyomugasho, Kamau, Aravindakshan, & Hendrickx, 2021) . Enzymes for simulated digestion were provided by Sigma Aldrich (Belgium): porcine pepsin (P7012, 3276.8 U/mg), porcine trypsin (T0303, 232.5 U/mg), bovine chymotrypsin (C4129, 55.8 U/mg), and porcine pancreatin (P1750, 30.72 U/mg amylase, 1.33 U/mg trypsin, and 0.94 U/mg chymotrypsin).
Activities of pure enzymes were provided by the manufacturer, while enzyme activity in pancreatin was determined as recommended in the INFOGEST protocol (Brodkorb et al., 2019) . All chemicals were of analytical or HPLC grade.
Activities of pure enzymes were provided by the manufacturer, while enzyme activity in pancreatin was determined as recommended in the INFOGEST protocol (Brodkorb et al., 2019) . All chemicals were of analytical or HPLC grade.
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