In vitro CA activity assay was determined according to the manufacturer’s protocol for hCAII (R&D Systems, USA) with slight modification. Candidate hCAII inhibitors were dissolved and diluted in 100% (v/v) DMSO. The compound solution was mixed with diluted enzyme solution in the wells of a 96-well plate. The 4-nitrophenyl acetate (4-NPA) (Sigma-Aldrich) solution was added to start the reaction and incubated at 25°C for 2 h, where the 4-NPA was hydrolyzed into 4-nitrophenol (4-NP) which was detected as the change in absorbance at 405 nm. The final concentration of 4-NPA and hCAII in the initial assay was 1 mM and 1 ng/μl, respectively. The enzyme inhibition was expressed as the IC50 (50% inhibition concentration), calculated by dose response curves with at least five concentrations. The CA inhibitor, acetazolamide (AZA) (Sigma-Aldrich) was used as a reference compound.
4 npa
Manufactured by Merck Group
Sourced in United States
4-NPA is a laboratory reagent used in chemical analysis. It is a colorimetric indicator that can be used to detect the presence of certain compounds. The core function of 4-NPA is to serve as a chemical indicator in analytical procedures.
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4 protocols using 4 npa
1
In vitro Carbonic Anhydrase Inhibition Assay
2
Enzymatic Activity Assay and ABPP of Erc1
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To determine the enzymatic activity of the purified Erc1 protein, 4-nitrophenyl α-L-arabinofuranoside (4NPA) (Sigma-Aldrich, Steinheim) was diluted in 0.1 M sodium acetate buffer (pH 4) to concentrations of 1 mM and 5 mM. The 4NPA substrate was incubated with 1.34 and 9 μM Erc1 and heat-inactivated recombinant protein at 40 °C. After 10 min incubation, the reaction was stopped by adding 150 μl 2% trisodium phosphate buffer (pH: 12). The absorption was measured at 400 nm. The commercially available AFASE from Aspergillus niger (Megazyme, Ireland) was used as a positive control.
For ABPP assay, 15 µl (80 µg ml−1 stock) of Erc1, Erc1M2x, and AFASE recombinant proteins were incubated in 300 mM sodium acetate buffer pH:6.0 for 30 min with and without 50 µM DL69 α-L-arabinofuranosidase specific inhibitor7 (link). Subsequently, 5 µM α-L-arabinofuranosidase specific ME868 probe was added to the reaction mixture and incubated for 2 h at room temperature at dark. The reaction was stopped with 6X SDS-loading dye and the probe was detected by scanning the in-gel fluorescence with Cy5 filter (Ex. 650 nm, Em. 670 nm). Protein of loaded samples was visualized via Sypro Ruby (Ex. 450 nm, Em. 610 nm).
For ABPP assay, 15 µl (80 µg ml−1 stock) of Erc1, Erc1M2x, and AFASE recombinant proteins were incubated in 300 mM sodium acetate buffer pH:6.0 for 30 min with and without 50 µM DL69 α-L-arabinofuranosidase specific inhibitor7 (link). Subsequently, 5 µM α-L-arabinofuranosidase specific ME868 probe was added to the reaction mixture and incubated for 2 h at room temperature at dark. The reaction was stopped with 6X SDS-loading dye and the probe was detected by scanning the in-gel fluorescence with Cy5 filter (Ex. 650 nm, Em. 670 nm). Protein of loaded samples was visualized via Sypro Ruby (Ex. 450 nm, Em. 610 nm).
3
Assessing Glutathione S-Transferase Detoxification
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As detoxifying enzymes, tau class GSTs have many substrates. We chose the typical substrates for GST detoxification, including 1-chloro-2,4-dinitrobenzene (CDNB), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-nitrobenzyl chloride (NBC), 1,2-dichloro-4-nitrobenzene (DCNB), and 4-nitrophenyl acetate (4-NPA) (Sigma-Aldrich Corp., St. Louis, MO, USA). For GOPX detoxification, we chose two substrates, cumene hydroperoxide (Cum-OOH) and ethacrynic acid (ECA) (Sigma-Aldrich), to test the effect of G- and H-site residues on substrate activity. The activities toward CDNB, NBC, DCNB, and ECA were measured as described by Habig et al. [33 (link)], the activity toward NBD-Cl was measured as described by Ricci et al. [34 (link)], and the activity toward Cum-OOH was measured as described by Edwards and Dixon [35 ]. The structures of substrates are shown in Table 1 . All reactions were carried out at 25 °C using 60 mM GSH (Sangon Biotech). Each reaction was repeated three times, and experimental results were based on the mean values of three independent experiments.
4
Screening of Carboxylesterase Inhibitors
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The recombinant purified CES2 and CES1 enzymes, and 4-NPA were purchased from Sigma-Aldrich with product IDs E0412, E0162, and N8130, respectively. Hesperetin, naringenin, and 2’, 4’-dihydroxychalcone were purchased form Fisher Scientific (>95% purity). All compounds were dissolved in DMSO at 10mM. In the screening assay, the compounds were diluted in 50mM HEPES and the final compound concentration in the reaction system was adjusted to 50μM. In the IC50 determination assay, selected compounds were diluted in 50mM HEPES to obtain ten final concentrations from 300μM to 0.005μM with 3 fold decrement. The assays were conducted at 300μl total volume in 96-well plates. Reactions consisted of the following: 100μL 50mM HEPES with 20 units of enzyme, 10μL compound solution (various concentrations), 2μL 300mM 4-NAP, and 188μL 50mM HEPES, pH 7.4. The compound and 4-NAP solution were added into the plate first and then the reaction was initiated by the addition of enzyme solution. After 15 minutes incubation at 25°C, absorbance was measured using a microplate reader at 405nm in FLUOstar Omega Microplate Reader. The CES1/CES2 inhibitor compound bis-(4-nitrophenyl) phosphate (BNPP) was used as positive control in all the assays.
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