Synthetic peptide cleavage was performed as previously described in [91 ] with slight modifications. Briefly, 5 µg/ml of each SPATES was added to 200 μl of three different pNA-conjugated oligopeptides: N-Succinyl-Ala-Ala-Ala-p-nitroanilide, N-Benzoyl-L-arginine 4-nitroanilide and N-succinyl-ala-ala-pro-phe-p-nitroanilide (Millipore Sigma) at 1 mM concentration in a buffer containing 0.2 M NaCl, 0.01 mMZnSO4, 0.1 M MOPS (3-(N-morpholino) propanesulfonic acid), pH 7.3 were incubated at 37°C for 3 h and absorbance readings were determined at 410 nm. Readings were normalized to the maximum absorbance of positive control. All reactions were performed in triplicate.
N succinyl ala ala pro phe p nitroanilide
Manufactured by Merck Group
Sourced in United States
N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide is a synthetic peptide substrate used for the colorimetric determination of proteolytic enzyme activity, particularly chymotrypsin-like serine proteases. The enzymatic cleavage of the peptide bond releases the p-nitroaniline chromophore, which can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
α chymotrypsin, by Merck Group (4 mentions)
Trypsin, by Merck Group (3 mentions)
Dithiothreitol (dtt), by Merck Group (3 mentions)
Chymotrypsin, by Merck Group (3 mentions)
Bovine trypsin, by Merck Group (2 mentions)
N p tosyl gly pro lys 4 nitroanilide acetate salt, by Merck Group (2 mentions)
N methoxysuccinyl ala ala pro val p nitroanilide, by Merck Group (2 mentions)
α chymotrypsin from bovine pancreas, by Merck Group (2 mentions)
Calf thymus dna, by Merck Group (2 mentions)
Trifluoroethanol, by Merck Group (2 mentions)
Imidazole, by Merck Group (2 mentions)
α chemotrypsin, by Merck Group (2 mentions)
Lithium chloride (licl), by Merck Group (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (2 mentions)
Reduced glutathione, by Merck Group (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Fxia chromogenic substrate s 2366, by Diapharma (1 mentions)
Thromboplastin d, by Thermo Fisher Scientific (1 mentions)
Aptt reagent, by Thermo Fisher Scientific (1 mentions)
Trypsin substrate s 2222, by Diapharma (1 mentions)
32 protocols using n succinyl ala ala pro phe p nitroanilide
1
Peptidase Activity Assay with Chromogenic Substrates
2
Coagulation Enzyme Assay Protocols
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Pentamidine isethionate was purchased from Millipore-Sigma (St. Louis, MO, USA). Reagents for clotting assays including thromboplastin-D, APTT reagent, and thrombin time reagent were all from Fisher Scientific (Pittsburgh, PA, USA). Chemicals used to prepare enzyme assay buffers were from Millipore-Sigma or Fisher Scientific. N,N–dimethylcasein, dansylcadaverine, and dithiothreitol were also from Millipore-Sigma. All types of plasmas were purchased from George King Bio-Medical, Inc. (Overland Park, KS, USA). Coagulation enzymes including thrombin, factor Xa (FXa), factor XIa (FXIa), and factor XIIIa (FXIIIa) were from Haematologic Technologies, Inc. (Essex Junction, VT, USA). Digestive enzymes including trypsin and chymotrypsin were from Millipore-Sigma. Neutrophil elastase was from Elastin Products Company (Owensville, MO, USA). Chromogenic substrates: Spectrozyme TH, Spectrozyme FXa, and Spectrozyme PL, were obtained from Biomedica-Diagnostics (Windsor, NS, Canada). FXIa chromogenic substrate (S-2366) and trypsin substrate (S-2222) were obtained from Diapharma (West Chester, OH, USA). Chromogenic substrate for chymotrypsin (N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and that for neutrophil elastase (S-1384) were from Millipore-Sigma.
3
Trypsin and Chymotrypsin Enzyme Inhibition Assay
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A possible inhibitor or enhancer of trypsin/chymotrypsin activity present in
venom fraction was tested. The reaction mixture was prepared as described by the
supplier (PBS, enzyme, venom and chromogenic substrate specific for each
enzyme). The assay was performed with bovine trypsin and α-chymotrypsin (1 mg/50
mL, 0.001 M HCl; Sigma-Aldrich Co., USA), trypsin and α-chymotrypsin substrates
(10 mg/mL N-p-Tosyl-Gly-Pro-Lys-4-nitroanilide acetate salt and
0.2 mg/mL N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide,
respectively; Sigma-Aldrich Co., USA), phosphate-buffered saline (PBS) pH 7.4,
and Fraction I at two concentrations (1.90 mg/mL and 5.65 mg/mL). The substrate
solution was prepared at the 1 mg/20 mL assay concentration in PBS. Volumes of 5
μL of bovine trypsin and α-chymotrypsin were tested with 5 μL of substrate in
the 96-well samples at room temperature with 100 μL PBS. The reaction mixtures
were read at 410 nm to quantify the formation of p-nitroaniline (yellowish
color) every 2 minutes during 200 minutes.
venom fraction was tested. The reaction mixture was prepared as described by the
supplier (PBS, enzyme, venom and chromogenic substrate specific for each
enzyme). The assay was performed with bovine trypsin and α-chymotrypsin (1 mg/50
mL, 0.001 M HCl; Sigma-Aldrich Co., USA), trypsin and α-chymotrypsin substrates
(10 mg/mL N-p-Tosyl-Gly-Pro-Lys-4-nitroanilide acetate salt and
0.2 mg/mL N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide,
respectively; Sigma-Aldrich Co., USA), phosphate-buffered saline (PBS) pH 7.4,
and Fraction I at two concentrations (1.90 mg/mL and 5.65 mg/mL). The substrate
solution was prepared at the 1 mg/20 mL assay concentration in PBS. Volumes of 5
μL of bovine trypsin and α-chymotrypsin were tested with 5 μL of substrate in
the 96-well samples at room temperature with 100 μL PBS. The reaction mixtures
were read at 410 nm to quantify the formation of p-nitroaniline (yellowish
color) every 2 minutes during 200 minutes.
4
Chymotrypsin Inhibition Assay Protocol
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Assay mixtures comprised 10 µL N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma-Aldrich, 200 µM final), 70 µL of reaction buffer (0.1 M Tris-HCl, 10 mM CaCl2, 250 mM NaCl, pH 8.0). Reactions were monitored by A405 nm at 30 °C after adding 10 µL of chymotrypsin (TLCK-treated, Sigma-Aldrich) in reaction buffer (2 nM final). Inhibitor was added in 10 µL aliquots, as above. For comparative studies using chymotrypsin (2 µg/mL) and Der p 1 (20 µg/mL), the above conditions were modified to permit the inclusion of 10 µL DTT (1 mM final) and in these experiments AEBSF (400 µM) or ADZ 50,000 (40 µM) were used as inhibitors.
5
Biofortification of Pearl Millet
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Bovine pancreatic trypsin, bovine pancreatic chymotrypsin, papain from papaya tree latex, and bromelain from pineapple stem were procured from Sisco Research Laboratory (Mumbai, India). Bovine serum albumin (BSA), ficin (fig tree latex), N-α-benzoyl-dl-arginine-p-nitroanilide (BApNA), N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAApNA), pGlu-Phe-Leu-p-nitroanilide (pFLNA), N-acetyl-d-glucosamine, laminarin, crab shell chitin, and dithiotheitol (DTT) were purchased from Sigma (St. Louis, MO). Glucose estimation kit procured from Kamineni Life Sciences, Hyderabad. X-ray films were purchased from Carestream Health India Pvt Ltd. (Mumbai). All other chemicals and reagents used were of analytical grade. 1). All these lines were biofortified using classical recombinant breeding approaches (Govindaraj et al. 2019a) . Briefly, biofortification was achieved by crossing among the high Fe/Zn lines and later to the elite lines followed by pedigree breeding to fix the line with high Fe/Zn in agronomically superior backgrounds at F 6 and later stages. These micronutrients are highly heritable and predominantly controlled by additive genes, and therefore, it is highly feasible to breed biofortified lines in pearl millet. Pearl millet biofortification breeding approaches are well described elsewhere (Rai et al. 2012; Govindaraj et al. 2013 Govindaraj et al. , 2019b)) .
6
Savinase Enzyme Assay Protocol
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Example 15
The substrate for the Savinase enzyme assay is the chromogenic peptide substrate N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma-Aldrich). This substrate is highly specific for subtilisin-like enzymes (Davis et al., 1999) and it can support enzyme assays in bacterium suspensions (Bonifait et al., 2010). In a typical assay, 100 μL of lysate, or bacterium suspension is added to 20 μl of the chromogenic substrate N-succinyl-Ala-Ala-Pro-Phe-pNa (2 mg/mL in 50% dimethyl formamide), the reaction mixture is incubated at 37° C. for variable times and the release of pNA is quantified by measuring the absorbance at 415 nm (Bonifait et al., 2010). This protocol is easily adaptable through automation to support screening by performing high throughput protease activity assays. Proteolytic activity can also be measured by digestion of AZO-casien (Vazquez et al. 2004). Twenty microliters of lysate are incubated in 384-well plate with 20 μL of 1% (w/v) AZO-casein in Tris-HCl buffer (0.1 M, pH8.0) and 0.5 mM CaCl2 at 55° C. for 30 min. After stopping the reaction with 40 μL of 5% (w/v) trichloracetic acid, reaction mixture is centrifuged and absorbance of supernatant was measured at 340 nm.
7
Neutrophil extracellular traps and serine protease activity
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NETs generated in response to PMA or RgpA were collected, and the activities of neutrophil serine proteases were measured using specific substrates. NE activity was assayed using N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (Sigma-Aldrich) as the substrate, while cat G activity was assayed using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma-Aldrich) as the substrate. The substrate [1 mM; in 100 μl of 50 mM Tris-HCl (pH 7.5)] was mixed with 100 μl of supernatant from the netting and control neutrophils, and the rate of substrate hydrolysis was measured as the increase in the optical density at 450 nm (OD405) after incubation for 30 min at 37°C.
8
Savinase Enzyme Assay Protocol
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Example 15
The substrate for the Savinase enzyme assay is the chromogenic peptide substrate N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma-Aldrich). This substrate is highly specific for subtilisin-like enzymes (Davis et al., 1999) and it can support enzyme assays in bacterium suspensions (Bonifait et al., 2010). In a typical assay, 100 μL of lysate, or bacterium suspension is added to 20 μl of the chromogenic substrate N-succinyl-Ala-Ala-Pro-Phe-pNa (2 mg/mL in 50% dimethyl formamide), the reaction mixture is incubated at 37° C. for variable times and the release of pNA is quantified by measuring the absorbance at 415 nm (Bonifait et al., 2010). This protocol is easily adaptable through automation to support screening by performing high throughput protease activity assays. Proteolytic activity can also be measured by digestion of AZO-casien (Vazquez et al. 2004). Twenty microliters of lysate are incubated in 384-well plate with 20 μL of 1% (w/v) AZO-casein in Tris-HCl buffer (0.1 M, pH8.0) and 0.5 mM CaCl2 at 55° C. for 30 min. After stopping the reaction with 40 μL of 5% (w/v) trichloracetic acid, reaction mixture is centrifuged and absorbance of supernatant was measured at 340 nm.
9
Kinetic Analysis of Subtilisin Enzyme Activity
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The activity of the subtilisin enzyme was tested in a solution-phase with a peptide (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) that, upon proteolysis, releases 4-nitroanaline, which absorbs light at wavelength of 410 nm. Initial substrate concentration was 0.8 mM with an enzyme concentration of 0.12 mg/L. The reaction was conducted at 37°C and a consistent pH of 7.8. pH was maintained by using a 50 mM Phosphate buffer. Activity was characterized with calculated values of KM and Vmax. Wavelength readings were obtained using the UV-6300PC Double Beam Spectrophotometer. Subtilisin Carlsberg was purchased from Sigma Aldrich (P5380) and N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide was purchased from Sigma Aldrich (S7388). 4-nitroanaline (to make standard curve for concentration analysis to test enzyme activity) was purchased from Millipore Sigma (185310).
10
Enzymatic Assay of Serine Proteases
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