Nα benzoyl l arginine 4 nitroanilide hydrochloride

Manufactured by Merck Group

Sourced in United States

Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride is a synthetic compound commonly used as a substrate for the protease enzyme trypsin. It is a white to off-white crystalline powder that is soluble in water and other polar solvents.

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10 protocols using nα benzoyl l arginine 4 nitroanilide hydrochloride

Proteomic Sample Preparation Reagents

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Hydrochloric acid (37%, AnalaR NORMAPUR),
sodium hydroxide (pellets, AnalaR NORMAPUR), acetonitrile (for UPLC/UHPLC
instruments, HiPerSolv CHROMANORM), and methanol (for UPLC/UHPLC instruments,
HiPerSolv CHROMANORM) were purchased from VWR International (Milan,
Italy). Sodium phosphate dibasic (pure) was purchased from Carlo Erba
(Cornaredo, Italy). Ammonium bicarbonate (≥99.5%) was purchased
from Fluka Analytical (St. Gallen, Switzerland). Urea (for synthesis)
was purchased from Merck Schuchardt (Hohenbrunn, Germany). TI from Glycine max (soybean, lyophilized powder, 13471 U/mg),
trypsin from porcine pancreas (lyophilized powder, 1000–2000
BAEE units/mg solid), α-chymotrypsin from bovine pancreas (type
II, lyophilized powder, >40 units/mg protein), DL-dithiothreitol
(≥99% titration), iodoacetamide (crystalline), calcium chloride
(anhydrous), formic acid (≥96%), and Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride (>98%, TLC) were purchased
from Sigma-Aldrich (St. Louis, MO, USA).

Prandi B., Vacca C., Sforza S, & Tedeschi T. (2023). Label-Free Quantification by Liquid Chromatography–Tandem Mass Spectrometry of the Kunitz Inhibitor of Trypsin KTI3 in Soy Products. Journal of Agricultural and Food Chemistry, 71(22), 8648-8655.

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Trypsin Inhibition Assay for Icariin Analogs

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Icariin and compounds 1–9 were assayed against bovine trypsin (Sigma-Aldrich, T1426) at 1x and 10x the IC₅₀ concentration. Compounds 3 and 7 were also assayed at 50x and 250x the IC₅₀ concentration. In assay buffer (50 mM Tris-HCl, 20 mM CaCl₂, pH 8.19 with or without 0.5% igepal), 0.002 mg freshly made trypsin is incubated with drugs for 5 minutes at 25 °C. 1 mM of substrate (Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride, Sigma-Aldrich, B3133) is added to start the reaction. Final reaction volume is 100 μl. Product formation is monitored by increasing absorbance at 410 nm at 25 °C for 30 minutes in kinetic mode using a SpectraMax M5 plate reader (Molecular Devices). All values were measured in duplicate.
To assay the effect of increasing PDE5 concentration on icariin and icariin analogs, the PDE5 inhibition assay described in the methods section was done with modifications. Compounds at approximately 1x the PDE5 IC₅₀ concentration were incubated with 1 or 2 nM PDE5, and GMP production was measured as described. All values were measured in triplicate.

Chau Y., Li F.S., Levsh O, & Weng J.K. (2019). Exploration of icariin analog structure space reveals key features driving potent inhibition of human phosphodiesterase-5. PLoS ONE, 14(9), e0222803.

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Michaelis-Menten Kinetics for Protease

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Michaelis-Menten constants (K_m) and turnover numbers (k_cat) were determined using L-BAPA (N_α-Benzoyl-L-arginine 4-nitroanilide hydrochloride, Sigma-Aldrich, Taufkirchen, Germany) as substrate. The enzyme concentration was 30 nM and the L-BAPA concentrations ranged from 0.02 mM to 9.5 mM. Assays were carried out in 20 mM Hepes pH 7.4, 150 mM NaCl, 2 mM CaCl₂ and 0.05% Tween 20. Using the linear portion of the curves, initial conversion rates were determined at 37 °C. The amount of active enzyme was determined by active site titration using SPINK1. Absorbance of L-BAPA was monitored at 405 nm using a Cytation5 microplate reader (BioTek, VT, USA).

Nagel F., Susemihl A., Geist N., Möhlis K., Palm G.J., Lammers M, & Delcea M. (2022). Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin. Journal of Inflammation Research, 15, 3633-3642.

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Trypsin Inhibition Assay Protocol

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Trypsin inhibition assays were done according to described methods [38 (link)], with following modifications: pH of the buffer solution (0.5 M Tris-HCl, 1 M NaCl, Sigma-Aldrich, St. Louis, MO, USA, and 0.01 M CaCl₂, Riedel-De-Haën, Seelze, Germany) was set to 7.5 and to completely dissolve the substrate N_α-benzoyl-L-arginine 4-nitroanilide hydrochloride (Sigma-Aldrich, St. Louis, MO, USA), 100% DMSO (1 mL) was added. Trypsin (Sigma-Aldrich, St. Louis, MO, USA) from porcine pancreas was used. The plate was incubated at 25 °C and the absorbance (405 nm) was read after 10 min. Six concentrations (10, 20, 30, 40, 50, and; 60 µg·mL⁻¹) of the inhibitor aprotinin (Sigma-Aldrich, St. Louis, MO, USA) in ultrapure water were used as positive control.

Häggqvist K., Toruńska-Sitarz A., Błaszczyk A., Mazur-Marzec H, & Meriluoto J. (2016). Morphologic, Phylogenetic and Chemical Characterization of a Brackish Colonial Picocyanobacterium (Coelosphaeriaceae) with Bioactive Properties. Toxins, 8(4), 108.

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DSS-Induced Intestinal Inflammation Assay

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UCB, N-benzoyl-L-tyrosine ethyl ester, and N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride were purchased from Sigma–Aldrich (St. Louis, MO, United States). DSS (36-50 kDa) was obtained from MP Biomedical (Solon, OH, United States). Enzyme linked immunosorbent assay (ELISA) kits for D-lactate, TNF-α, IL-1β, and myeloperoxidase (MPO) were from Beijing Propbs Biotechnology (Beijing, China). The antibodies used in this study were anti-TLR4 (19811-1-AP; Proteintech, Rosemont, United States), anti-MyD88 (4283; Cell Signaling Technology, Danvers, MA, United States), anti-TRAF6 (ab33915; Abcam, Cambridge, MA, United States), anti-inhibitor of NF-κB alpha (IκBα) (4814; Cell Signaling Technology), and anti-occludin (ab167161; Abcam). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), goat anti-rabbit immunoglobulin G and goat anti-mouse immunoglobulin G were purchased from ZSGB-BIO Co. Ltd. (Beijing, China). All other reagents used were of analytical grade.

Zheng J.D., He Y., Yu H.Y., Liu Y.L., Ge Y.X., Li X.T., Li X., Wang Y., Guo M.R., Qu Y.L., Qin X.F., Jiang M.S, & Wang X.H. (2019). Unconjugated bilirubin alleviates experimental ulcerative colitis by regulating intestinal barrier function and immune inflammation. World Journal of Gastroenterology, 25(15), 1865-1878.

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MALDI-TOF Mass Spectrometry of Rat Brain

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Acetonitrile (ACN), trifluoroacetic acid (TFA), CHCA, diisopropylethylamine (DIEA), trypsin from bovine pancreas, cytochrome c from equine heart, n_α-benzoyl-L-arginine 4-nitroanilide hydrochloride, triethanolamine, calcium chloride, sodium chloride and hydrochloric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. Conductive indium tin oxide (ITO)-coated microscope slides were purchased from Delta Technologies (Loveland, CO, USA). Frozen rat brain was obtained from Pel-Freez (Rogers, AR, USA) and stored at −80 °C.

Zubair F., Laibinis P.E., Swisher W.G., Yang J., Spraggins J.M., Norris J.L, & Caprioli R.M. (2016). Trypsin and MALDI matrix pre-coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry. Journal of mass spectrometry : JMS, 51(12), 1168-1179.

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Isolation and Characterization of Bioactive Compounds

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The extraction and isolation solvents were purchased from Junsei Chemical Co., Ltd., Tokyo, Japan. Acetonitrile used for LC analyses was obtained from Fisher Scientific company, Hampton, NH, USA. Iodine solution, acarbose, and γ-oryzanol were procured from Fujifilm Wako Pure Chemical Corporation, Osaka, Japan. Silica gel, α-amylase from porcine pancreas (type VI-B), α-glucosidase from Saccharomyces cerevisiae, trypsin from porcine pancreas, soluble starch, p-nitrophenyl-α-D-glucopyranoside (pNPG), Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) and all buffer components were acquired from Sigma-Aldrich, St. Louis, MO, USA.

Quan N.V., Xuan T.D., Tran H.D., Ahmad A., Khanh T.D, & Dat T.D. (2019). Contribution of momilactones A and B to diabetes inhibitory potential of rice bran: Evidence from in vitro assays. Saudi Pharmaceutical Journal : SPJ, 27(5), 643-649.

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Quantitative Protease Activity Assay

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Different concentrations of each recombinant protein were incubated with three different serine proteases in Tris buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.1% BSA; pH 7.4): factor Xa (FXa) (5 × 10⁻⁵ U), thrombin (2.5 × 10⁻³ U), or trypsin (9 U) (Sigma-Aldrich®) at 37°C for 15 min in 0.5 ml microtubes. Then, the tube contents were placed in 96-well plates and the enzymatic reactions were triggered in the presence of the specific substrates CH₃OCO-D-CHA-GlyArg-pNA-AcOH (Sigma-Aldrich®) for FXa, Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride (Sigma-Aldrich®) for trypsin, and N-(p-tosyl)-Gly-Pro-Arg p-nitroanilide acetate salt (Sigma-Aldrich®) for thrombin at 4 mM concentration and 100 µl final volume. Immediately after the addition of the substrate, the absorbance variation was assessed at 405 nm every 10 s with 2 s agitation between reads in a microplate reader (VersaMax™, Molecular Devices) for 30 min. The maximum reaction velocity (Vmax) values were obtained for reactions lacking inhibitors and were used as a reference for reactions in the presence of different concentrations of inhibitors.

Costa G.C., Ribeiro I.C., Melo-Junior O., Gontijo N.F., Sant’Anna M.R., Pereira M.H., Pessoa G.C., Koerich L.B., Oliveira F., Valenzuela J.G., Giunchetti R.C., Fujiwara R.T., Bartholomeu D.C, & Araujo R.N. (2021). Amblyomma sculptum Salivary Protease Inhibitors as Potential Anti-Tick Vaccines. Frontiers in Immunology, 11, 611104.

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Serine Protease Inhibition Assay

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The inhibitory activities of 8 rCvT-serpins were determined using 4 serine proteases, including trypsin (Sigma, USA), α-chymotrypsin (Sigma, USA), elastase (Sigma, USA), and subtilisin A from B. licheniformis (Sigma, USA), as previously depicted with wispy modifications (Gu et al. 2021 (link)). The residual protease activities were detected at 405 nm on a microplate reader (Thermo Fisher Scientific, USA) by the addition of 1 mM specific chromogenic substrates in 50 mM Tris-HCl buffer containing 50 mM NaCl and 5 mM CaCl₂, pH 7.5, Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride (Sigma B3133, USA) for trypsin, N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma S7388, USA) for α-chymotrypsin, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide (Sigma S8511, USA) for elastase, and Z-Gly-Gly-Leu p-nitroanilide (Sigma C3022, USA) for subtilisin A.

Wu Z., Yuan R., Gu Q., Wu X., Gu L., Ye X., Zhou Y., Huang J., Wang Z, & Chen X. (2023). Parasitoid Serpins Evolve Novel Functions to Manipulate Host Homeostasis. Molecular Biology and Evolution, 40(12), msad269.

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Enzymatic Assay Substrate Preparation

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Substrates (3-methylcatechol and N_α-Benzoyl-l-arginine
4-nitroanilide hydrochloride), buffer salts (HEPES, Tris-HCl, and
Na₂HPO₄/NaH₂PO₄), and
metal salts (MnCl₂·4H₂O) were obtained
from Sigma-Aldrich (St. Louis, MO, United States).

Forero N., Liu C., Sabbah S.G., Loewen M.C, & Yang T.C. (2023). Assay Development for Metal-Dependent Enzymes—Influence of Reaction Buffers on Activities and Kinetic Characteristics. ACS Omega, 8(43), 40119-40127.

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