This analysis, described by Brodkorb et al. (2019 (link)), was carried out to assess the activity of the trypsin present in the pancreatin used in the intestinal phase of the in vitro digestion. For this assay, 10 mM p-toluene-sulfonyl-l -arginine-methyl-ester (TAME) (Sigma-Aldrich) was used as trypsin substrate. The reaction of TAME in the presence of trypsin produces p-toluene-sulfonyl-l -arginine and methanol. The objective of the test was to measure the absorbance increase over time to determine the hydrolytic activity of trypsin per min. For this, a solution of trypsin at different concentrations: 10 µg/mL, 15 µg/mL and 20 µg/mL in 1 mM HCl and 46 mM Tris–HCl buffer with 11.5 mM CaCl2, pH 8.1, were prepared. First, 2.6 mL of Tris–HCl buffer was mixed with 300 µL of TAME and 100 µL of trypsin and then, continuous absorbance measurement was performed at 247 nm for 10 min. To measure the absorbance, the model 6505 UV/Vis spectrophotometer (Jenway, Stone, UK) was used. Subsequently, the same test was carried out with the pancreatin (1 mg/mL) used in the GIT digestion. The amount of pancreatin used in the digestion assays was based on the activity of the trypsin present in it.
P toluene sulfonyl l arginine methyl ester tame
Manufactured by Merck Group
Sourced in United States
P-toluene-sulfonyl-l-arginine-methyl-ester (TAME) is a chemical compound used as a substrate in various enzymatic assays. It is commonly employed to measure the activity of proteolytic enzymes, particularly those that cleave peptide bonds involving the arginine amino acid residue.
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Lab products found in correlation
Glycerol, by Merck Group (1 mentions)
Tris hydroxymethyl aminomethane hydrochloride tris hcl, by Merck Group (1 mentions)
Bovine bile, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Alexa fluor 594 conjugated wheat germ agglutinin, by Thermo Fisher Scientific (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Calcium chloride dihydrate, by Merck Group (1 mentions)
Porcine pancreatin, by Merck Group (1 mentions)
Monopotassium phosphate, by Merck Group (1 mentions)
Ammonium carbonate, by Merck Group (1 mentions)
Magnesium dichloride hexahydrate, by Merck Group (1 mentions)
Laemmli sample buffer 2, by Bio-Rad (1 mentions)
Cumene hydroperoxide, by Merck Group (1 mentions)
Magnesium chloride mgcl2, by Merck Group (1 mentions)
Bile bovine dried, by Merck Group (1 mentions)
4 protocols using p toluene sulfonyl l arginine methyl ester tame
1
Trypsin Activity Measurement in Pancreatin
2
In Vitro Digestion Protocol with Simulated Fluids
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All the reagents used in this study were of analytical or higher grade. Sodium phosphate, ammonium bicarbonate (AmBic), acetic acid and the other chemicals used to produce the simulated salivary fluid (SSF), the simulated gastric fluid (SGF) and the simulated intestinal fluid (SIF), were provided by Carlo Erba (Milan, Italy). The enzymes used for in vitro human digestion were purchased from Sigma (St Louis, MO, USA), in line with those recommended by the INFOGEST protocol [33 (link)]. Trichloroacetic acid (TCA), sodium dodecyl sulfate (SDS), glycerol, tris(hydroxymethyl) aminomethane hydrochloride (Tris-HCl), ethylenediaminetetraacetic acid (EDTA), guanidine chloride, trifluoroacetic acid (TFA), formic acid (FA), acetonitrile (ACN), 2-vinylpyridine monomer, and p-toluenesulfonyl-l -arginine methyl ester (TAME) were also from Sigma-Aldrich. Egg lecithin was purchased from Lipid Products (Redhill, UK). The electrophoresis reagents were all obtained from Bio-Rad (Milan, Italy).
The protein-free bread (<0.1%, w/w) (Amino’ pane le rosette produced by Antica Farmacia Orlandi) was purchased from a local pharmacy.
The protein-free bread (<0.1%, w/w) (Amino’ pane le rosette produced by Antica Farmacia Orlandi) was purchased from a local pharmacy.
3
Protein Extraction and Separation Protocol
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Phosphate-buffered saline (PBS), paraformaldehyde, potassium chloride, sodium bicarbonate, sodium chloride, monopotassium phosphate, magnesium dichloride hexahydrate, ammonium carbonate, calcium chloride dihydrate, hydrochloric acid, bovine bile, porcine pepsin, porcine pancreatin and p-toluene-sulfonyl-L-arginine methyl ester (TAME) were obtained from Merck (Amsterdam, Netherlands). Laemmli sample buffer (2 ×) was obtained from Bio-Rad (Lunteren, The Netherlands). Non-heat inactivated non-irradiated fetal bovine calf serum(FBS) with product number FBS-12a was obtained from Capricorn Scientific (Ebsdorfergrund, Germany). Trifluoroacetic acid, dithiothreitol and iodoacetamide were obtained from Sigma Aldrich (Zwijndrecht, The Netherlands), and sequencing grade trypsin was obtained from Boehringer Mannheim (Mannheim, Germany). Precast SurePAGE MOPS-Tris PAGE gels were obtained from Genscript (Rijswijk, The Netherlands). Alexa-fluor 594-conjugated wheat germ agglutinin was obtained from Thermo Fisher (Bremen, Germany).
4
Quantifying Omega-3 Fatty Acids in Fish Oils
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Bile bovine dried, cumene hydroperoxide, p-toluene-sulfonyl-L-arginine methyl ester (TAME), glyceryl tributyrate (tributyrin), sodium taurodeoxycholate, Tris-(hydroxymethyl)-aminomethane (Tris), tetraethoxypropane (TEP), potassium chloride (KCl), sodium bicarbonate (NaHCO3), calcium chloride (CaCl2), and magnesium chloride (MgCl2) were purchased from Merck & Co. (New Jersey, NJ, USA). Sodium chloride (NaCl), potassium dihydrogen phosphate (KH2PO4), hydrochloric acid 37% (HCl), chloroform (CHCl3), methanol (CH3OH), ammonium thiocyanate (NH4SCN), and iron (III) chloride (FeCl3) were purchased from Chem-Lab NV (Zedelgem, Belgium). Porcine pepsin, pancreatin, and ammonium carbonate (NH4)2CO3 were purchased from Central Drug House Ltd. (New Delhi, India). Butylated hydroxytoluene (BHT) and -thiobarbituric acid (TCA) were purchased from Fluorochem Ltd. (Hadfield, UK). Sodium hydroxide (NaOH) and trichloroacetic acid (TCA) were purchased from Lach-Ner Ltd. (Neratovice, Czech Republic). All the chemicals and reagents used in this study were of analytical or HPLC grade. The samples tested were various well-known brands of fish oil chewable tablets and capsules purchased from local pharmacies; fresh sardines that were purchased from local vendor; and eggs rich in omega-3 fatty acids, which were kindly offered by Avgodiatrofiki S.A. (Nea Santa, Kilkis, Greece).
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