N methoxysuccinyl ala ala pro val p nitroanilide

Neutrophil elastase activity was analysed with the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma). The baso-lateral cell medium from ALI-cultures was diluted 1:4 in HEPES buffer (0.1 M HEPES pH 7.5 + 0.5 M NaCl). Fresh cell medium was used as a control. Equal amounts of diluted cell medium and 0.05 U/ml neutrophil elastase in HEPES buffer (40 μl each) (Sigma, St. Louis, MO, USA) were mixed in a 96-well plate. Samples were incubated at room temperature for 20 min. To each well, 100 μl of 0.2 mM N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide in HEPES buffer was added. The plate was incubated at room temperature, and the absorbance was determined at 405 nm at different time points.

Twenty milliliters of urine from each time point was centrifuged to separate cells and extracellular components. Mitochondrial DNA (mtDNA) concentrations within the urine supernatants were assessed by real time quantitative polymerase chain reaction (RT-qPCR) for swine mitochondrial-encoded cytochrome b (Mt-cyb), and compared against a standard curve of known mtDNA concentrations (Bio-Rad Laboratories Inc, Hercules, CA, USA). Urine supernatant samples were adjusted to between 6.5–7.5 pH with 10 N NaOH.
Concentrations of swine inflammatory markers IL-1β, IL-6, IL-8, IL-10, and TNF-α in 25 μL of urine were measured by LuminexTM xMAP technology using porcine ProcartaPlexTM Simplex Kits (Life Technologies Inc, Carlsbad, CA, USA) against a standard curve of known concentrations for each marker. The amount of NaOH was negligible and thus not factored into the dilution. Activity of secreted neutrophil elastase (NE) in urine supernatants was measured by hydrolysis of the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma Aldrich Inc, St. Louis, MO, US) into 4-nitroaniline after incubating for 1 hour at 37 °C + 5% CO₂ by light absorbance at 405 nm via spectrophotometry with a NanoDrop 2000 (Thermo Fisher Scientific).

Ten μl of liquefied sputum sample was mixed with 90 μl of HEPES buffer (0.1 M HEPES, 0.5 M NaCl; pH 7.5), supplemented with 5 mM EDTA, 2 μg/ml of E64 (Sigma, St. Louis, MO) and 1 μg/ml of Pepstatin A (Sigma-Aldrich, St. Louis, MO) in a 96-wells plate. Neutrophil elastase (Sigma-Aldrich) in 2-fold dilutions from a 2 U/ml stem solution was used as a standard. Fifty μl of the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) was added to each well to a final concentration of 0.5 mM. The plates were incubated at 37 °C and the absorbance at 415 nm was determined every 10 min to measure enzyme kinetics. All samples were analysed in duplicates.

Elastase enzyme activity in NET samples was measured using an enzymatic colorimetric method with the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich). NET solution and 1 mM elastase substrate were mixed. After 30 min, absorbance was measured at 405nm in a microplate reader. Various concentrations of purified elastase enzyme from human neutrophils (EMD Chemicals Inc., Billerica, MA, USA) were used as standards.

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.

5 μM human α1-antitrypsin (Sigma) or human antithrombin (Aclotin^®, LFB) were incubated with 50 μM annonacinone (or 0.5% DMSO in reaction buffer as control) for 20 min at 37 °C. The mixtures containing α1-antitrypsin were diluted in N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma) containing solution and substrate hydrolysis kinetic was triggered by addition of neutrophil elastase (Sigma) to a final volume of 100 μL containing 13 nM α1-antitrypsin, 130 nM annonacinone, 82 μM chromogenic substrate and 5 nM neutrophil elastase. The mixtures containing antithrombin were diluted in fondaparinux (Arixtra^®, GlaxoSmithKline) and CS-11(22)-FXa (Hyphen Biomed) containing solution and substrate hydrolysis kinetic was triggered by addition of factor Xa (Stago BNL) to a final volume of 100 μL containing 51 nM antithrombin, 510 nM annonacinone, 200 μM chromogenic substrate, 5 μM fondaparinux and 2 nM factor Xa.

Gallic acid and folin ciocalteu as well as chemicals for ORAC assay such as sodium fluorescein, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®), Nunc Micro-well™ plates, phosphate buffer (pH 7.4) were purchased from Sigma Aldrich. ORAC experiment was performed on fluorometer, FLUOstar OPTIMA, Franka Ganske, BMG LABTECH, Offenburg, Germany. DPPH free radical solution, vitamin C (ascorbic acid), mushroom tyrosinase enzyme obtained from mushroom, l-3,4-dihydroxyphenylalanine (l-DOPA), potassium dihydrogen orthophosphate (pH 6.5), potassium hydroxide, hydroquinone monomethyl ether, elastase enzyme, substrate: (N-methoxy-succinyl-Ala-Ala-Pro-Val-P-nitro-anilide), tris(hydroxymethyl)-methyl-2-aminoethane sulfonate (TES) buffer, HEPES buffer which is abbreviation for (4-2-hydroxyethyl-l-piperazine ethane sulfonic acid), pH = 7.5, elafin positive control, collagenase type 1 from Clostridium histolyticum, FALGPA substrate (N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala) and epigallocatechin gallate (EGCG) were purchased from Sigma Aldrich. UV recordings were made on ELX 808 (Bio Tek Instrumental, Italy), plates were purchased from (Mekkawy, Egypt).