N succinyl ala ala pro phe p nitroanilide suc aapf pna

Granzyme activities were determined using colorimetric methods. Reaction buffer [50 mM Tris–HCl, 0.15 M NaCl, 0.01% Triton X-100 (pH 7.6) containing 0.2 mM Ellman's reagent (Sigma-Aldrich)] was added to CMC samples and cells lysed with extensive vortexing. Samples consisting on target cells or HKLs alone were used as controls. For this, 10 μL of samples were dispensed in 96-well plates and mixed with 90 μL of reaction buffer containing 0.2 mM of the corresponding specific substrates: Z-Lys-SBzl (Sigma-Aldrich) for tryptase/GzmA/K, N-Acetyl-Ile-Glu-Pro-Asp-p-nitroanilide (Ac-IEPD-pNA; Sigma) for aspartase/GzmB, Boc-Ala-Ala-Met-SBzl (MP Biochemicals) for metase/GzmM or N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA; Sigma) for chymase/GzmH activities. Substrates were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. Optical density (OD) was determined at 405 nm at 0 and 90 min of incubation being the OD change determined. Reaction buffer replaced the sample in blanks.

Nutrient broth and chemicals: Coomassie brilliant blue (CBB) R-250, 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris), Bradford reagent, bovine serum albumin (BSA) and acrylamide/bis-acrylamide solution were purchased from HiMedia (Mumbai, India). Low-molecular-mass marker was purchased from GeneDireX (Taoyuan, Taiwan). N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA), azocasein, gelatin, 4-aminobenzamidine dihydrochloride, dithiothreitol, sodium dodecyl sulfate (SDS), glycerol, bromophenol blue, trichloroacetic acid (TCA), ethylenediaminetetraacetic acid (EDTA), Triton X-100, ammonium sulfate, sodium acetate, sodium phosphate, glycine, NaCl, NaOH, HCl, KCl, CaCl₂, CuCl₂, ZnCl₂ and MgCl₂ were purchased from Sigma-Aldrich, Merck (St. Louis, MO, USA).

The proteolytic activities of culture supernatants of Natrinema sp. J7-2 and its derivatives were assayed using azocasein (Sigma, St. Louis, MO, United States) as the substrate. azocaseinolytic activity was determined at 37°C for 60 min in 200 μl reaction mixture containing 0.25% (w/v) azocasein and 100 μl culture supernatant in buffer A (50 mM Tris-HCl, 10 mM CaCl₂, 3 M NaCl, pH 8.0). The reaction was terminated by adding 200 μl 40% (w/v) trichloroacetic acid (TCA) into the reaction mixture. After incubation at room temperature for 15 min, the sample was centrifuged at 13,400 ×g for 10 min, and the absorbance of the supernatant at 335 nm (A₃₃₅) was measured in a 1 cm cell. One unit (U) of azocaseinolytic activity was defined as the amount of enzyme required to increase the A₃₃₅ by 0.01 per min under the assay conditions used.
The synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (suc-AAPF-pNA) (Sigma) was used to determine intracellular proteolytic activity. The cells of Natrinema sp. J7-2 and its derivatives were washed twice with buffer A and sonicated on ice in the same buffer. Subsequently, the cell extract was separated from the cell debris by centrifugation at 13,400 ×g for 10 min at 4°C and used to determine the proteolytic activity on 0.5 mM suc-AAPF-pNA at 37°C as described previously (Du et al., 2015 (link)).