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4 nitrophenyl acetate

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4-nitrophenyl acetate is a chemical compound commonly used as a laboratory reagent. It serves as a substrate for various enzymatic reactions, particularly those involving esterases and other hydrolytic enzymes. The compound's core function is to provide a measurable indicator of enzyme activity through the release of a colored product upon enzymatic hydrolysis.

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Lab products found in correlation

1 chloro 2 4 dinitrobenzene, by Merck Group (3 mentions) 4 nitrophenol, by Merck Group (2 mentions) 1 2 dichloro 4 nitrobenzene, by Merck Group (2 mentions) 1 phenyl 1 2 ethanediol, by Merck Group (2 mentions) Reduced l glutathione gsh and oxidized glutathione, by Carl Roth (2 mentions) 4 nitrophenyl butyrate, by Merck Group (2 mentions) 5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (2 mentions) Acetylthiocholine iodide, by Merck Group (2 mentions) Ethylenediaminetetraacetic acid edta, by Merck Group (2 mentions) Direct q 3 uv water purification system, by Merck Group (2 mentions) Advanced vortex mixer zx3, by VELP Scientifica (2 mentions) Trizma base, by Merck Group (2 mentions) 4 nitrophenyl substrate, by Merck Group (1 mentions) 4 nitrophenyl trimethylacetate, by Merck Group (1 mentions) Tri reagent, by Merck Group (1 mentions) Qubit double stranded dna dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions) Bradford reagent, by Merck Group (1 mentions) Avicel, by Merck Group (1 mentions) Luria bertani broth, by HiMedia (1 mentions) Cm cellulose, by Merck Group (1 mentions)

23 protocols using 4 nitrophenyl acetate

1

Measuring SIAE Enzymatic Activity

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The enzymatic activity of SIAE of transfected cells was determined using the general esterase artificial substrate 4-nitrophenyl acetate (pNPA) (Sigma), the release of the product 4-nitrophenol (pNP) (Sigma) can be measured at 405 nm, as described28 (link). Cells were seeded at 2,500 cells per well in 100 μL clear media in 96 well plates, in the presence and absence of doxycycline. Firstly, a 20 mM stock of pNPA was prepared in DMSO. Media was removed and the cells were washed twice with PBS. In each well, 220ul of Phosphate buffer (50 mM, pH 7.4) was added, pNPA was added using on board injectors to a final concentration of 0.1 mM or 0.2 mM and absorbance was measured at 410 nm using BMG OPTIMA PolarStar. A standard curve was generated using pNP (0.025–0.2 mM).
Mather R.L., Loveson K.F, & Fillmore H.L. (2019). Human Sialic acid O-acetyl esterase (SIAE) – mediated changes in sensitivity to etoposide in a medulloblastoma cell line. Scientific Reports, 9, 8609.
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2

Recombinant Expression of GST Enzymes

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Styrene oxide (racemic), 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrophenyl acetate (NPA), and 1-phenyl-1,2-ethanediol were purchased from Sigma-Aldrich (Steinheim, Germany). Reduced l-glutathione (GSH) and oxidized glutathione (GSSG) were ordered from Carl Roth (Karlsruhe, Germany).
The plasmids pET16bp_StyI and pET16bp_StyJ for the recombinant expression of the glutathione S-transferases carry the respective genes in a codon-optimized form synthesized by Eurofins MWG (NCBI GenBank accession numbers are MW590822 for styI and MW590823 for styJ). They were inserted into the multiple-cloning site (MCS) of pET16bp via the NdeI and NotI restriction sites, as described earlier for other genes (22 (link)). Correct insertion of constructs was checked via sequencing. Resulting expression products will carry an N-terminal His10 tag.
Lienkamp A.C., Burnik J., Heine T., Hofmann E, & Tischler D. (2021). Characterization of the Glutathione S-Transferases Involved in Styrene Degradation in Gordonia rubripertincta CWB2. Microbiology Spectrum, 9(1), 10.1128/spectrum.00474-21.
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3

Kinetic Analysis of 4-Nitrophenyl Substrates

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4-nitrophenyl substrate-specific activity was determined in 50 µL reactions containing 25 mM Tris pH 7.5, 250 mM NaCl, 1 mM MnCl2, 10% glycerol, 1 µM protein, and 1 mM 4-nitrophenyl substrate. The tested substrates, 4-nitrophenyl acetate (Sigma, N8130), 4-nitrophenyl butyrate (Sigma N9876), and 4-nitrophenyl trimethylacetate (Sigma 135046), were resuspended in acetonitrile at 100 mM. Reactions without 4-nitrophenyl substrate were preincubated at 37°C for 10 min prior to assay initiation via substrate addition. Conversion of 4-nitrophenyl substrates to 4-nitrophenol was tracked photometrically at 37°C and A405nm. Experiments were performed in triplicate with technical duplicates.
Miller J.J., Shah I.T., Hatten J., Barekatain Y., Mueller E.A., Moustafa A.M., Edwards R.L., Dowd C.S., Hoops G.C., Johnson R.J., Planet P.J., Muller F.L., Jez J.M, & Odom John A.R. (2021). Structure-guided microbial targeting of antistaphylococcal prodrugs. eLife, 10, e66657.
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4

Isolation and Characterization of Paenibacillus sp. LS1

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Paenibacillus sp. LS1 was isolated from a decaying wood sample collected from Lachung village, North Sikkim district (27°40′13″N, 88°43′37″E), Sikkim, India (7 (link)). Beechwood and corncob xylan were procured from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Avicel, CM-cellulose, 4-O-methyl-d-glucurono-d-xylan, and 4-nitrophenyl acetate were procured from Sigma-Aldrich, USA. All the chemicals required for minimal medium, Luria-Bertani broth, and 3,5-dinitrosalicylic acid (DNS) reagent preparation were procured from HiMedia Laboratories, Mumbai, India, unless otherwise specified. Bradford reagent and TRI reagent were procured from Sigma-Aldrich, USA. The Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit was procured from Thermo Fisher Scientific, USA.
Mukherjee S., Lodha T.D, & Madhuprakash J. (2023). Comprehensive Genome Analysis of Cellulose and Xylan-Active CAZymes from the Genus Paenibacillus: Special Emphasis on the Novel Xylanolytic Paenibacillus sp. LS1. Microbiology Spectrum, 11(3), e05028-22.
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5

Quantification of CPT, Irinotecan, and Metabolites

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Camptothecin (CPT; internal standard (I.S)), Irinotecan hydrochloride (Cat # I1406), Paclitaxel (Cat # T7402) and 4-Nitrophenyl acetate (PNPA; Cat # N8130) were purchased from Sigma-Aldrich, St. Louis, MO. SN-38 and SN-38G were kind gifts from Dr. Ming Hu’s laboratory at the University of Houston, Houston, TX. The sequences of the primers and probes were reported in our previous publications (Ghose et al., 2007 (link); 2008 (link); 2009 (link)). Bicinchoninic acid (BCA) assay kit (Cat # 23225) was purchased from Thermo Fisher Scientific Inc. All other reagents for real-time PCR were purchased from Applied Biosystems (Foster City, CA). Liquid chromatography mass spectrometry (LCMS) grade solvents were purchased from VWR international, LLC (Suwanee, GA, USA) for chromatography. Unless specified, all other materials were purchased from Sigma-Aldrich (St Louis, MO, USA.).
Mallick P., Basu S., Moorthy B, & Ghose R. (2017). Role of Toll-like receptor 4 in Drug-drug Interaction between Paclitaxel and Irinotecan in vitro. Toxicology in vitro : an international journal published in association with BIBRA, 41, 75-82.
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6

Carbonic Anhydrase II Activity Assay

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50 μl of cell lysate were mixed with CA II reaction buffer containing 12.5 mM TRIS pH 7.5, 75 mM NaCl and 2 mM 4-nitrophenylacetate (50 μl, Sigma). Absorbance was monitored at 400 nm for 5 min. Conversion to 4-nitrophenol was calculated from the slope of the absorbance plot using a calibration line from different dilutions of 1 mM 4-nitrophenol (Sigma).
Bernhardt A., Schamel M., Gbureck U, & Gelinsky M. (2017). Osteoclastic differentiation and resorption is modulated by bioactive metal ions Co2+, Cu2+ and Cr3+ incorporated into calcium phosphate bone cements. PLoS ONE, 12(8), e0182109.
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7

Gut Metagenome Enzyme Screening

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The slug gut metagenome DNA was provided by Dr Natalie Ferry, University of Salford, Manchester, United Kingdom. The substrates—4-nitrophenyl acetate, 4-nitrophenyl butyrate, 4-nitrophenyl octanoate, 4-nitrophenyl decanoate, and 4-nitrophenyl palmitate, β-D-glucose pentaacetate, pectin, chlorogenic acid, benzyl cinnamate, and 4-methylumbelliferyl acetate—were purchased from Sigma-Aldrich (Gillingham, United Kingdom). Xylan (birchwood, partially acetylated) was purchased from Megazyme (Wicklow, Ireland). Other chemicals were of molecular grade and purchased from Sigma-Aldrich (Gillingham, United Kingdom). Primers for PCR were synthesised and sequenced by Eurofins (Konstanz, Germany). BL21 and TOP10 competent cells were purchased from ThermoFisher Scientific (Leicester, United Kingdom).
Madubuike H, & Ferry N. (2022). Characterisation of a Novel Acetyl Xylan Esterase (BaAXE) Screened from the Gut Microbiota of the Common Black Slug (Arion ater). Molecules, 27(9), 2999.
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8

Purification and Characterization of MBP

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His-tagged maltose-binding protein (MBP) was expressed from a plasmid in E. coli and then purified through Ni-chelated Sepharose Fast Flow Resin (GE Healthcare) and HiLoad 16/60 Superdex 75 gel filtration column (GE Healthcare). The protein was exchanged into assay buffer (120 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 7.4) by dialysis. Both carbonic anhydrases were obtained from a commercial vendor (h-CA I (Sigma C4396) and b-CA II (Sigma C2522)). All protein concentrations were determined with Quick StartTM Bradford Protein Assay Kit (Bio-Rad, catalog no. 5000201).
Ligands were all obtained from commercial vendors, as follows: maltose (EMD Millipore 105910), acetazolamide (Sigma 97582), methazolamide (Sigma SML0720), sulfanilamide (Sigma 46874), trifluoromethanesulfonamide (Sigma 638455), and 4-nitrophenyl acetate (Sigma N8130).
Bai N., Roder H., Dickson A, & Karanicolas J. (2019). Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities. Scientific Reports, 9, 2650.
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9

Spectroscopic Characterization of Organic Compounds

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All reagents and solvents were of reagent grade quality and were obtained from commercial suppliers. All solvents were dried and purified as described by Perrin and Armarego.26 Sulphanilamide, Sepharose 4B, protein assay reagents, 4-nitrophenylacetate were obtained from Sigma-Aldrich Co. All other chemicals were analytical grade and obtained from Merck.
The IR spectra were recorded on a Perkin Elmer 1600 FT-IR spectrophotometer, using KBr pellets. 1H and 13C-NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometers in CDCl3 and chemical shifts were reported (δ) relative to Me4Si as internal standard. MALDI-MS of complexes were obtained in dihydroxybenzoic acid as the MALDI matrix, using a nitrogen laser accumulating 50 laser shots, with a Bruker Microflex LT MALDI-TOF mass spectrometer. Optical spectra in the UV-Vis region were recorded with a Perkin Elmer Lambda 25 spectrophotometer.
Arslan T., Biyiklioglu Z, & Şentürk M. (2018). The synthesis of axially disubstituted silicon phthalocyanines, their quaternized derivatives and first inhibitory effect on human cytosolic carbonic anhydrase isozymes hCA I and II. RSC Advances, 8(19), 10172-10178.
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10

Human Carbonic Anhydrase Enzyme Purification

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In this study, hCA
I and II were obtained from fresh human erythrocytes using affinity
chromatography as previously described in the literature.36 (link),37 (link) AChE from Electrophorus electricus (electric eel) (C3389: Sigma-Aldrich), BChE from equine serum (C4290:
Sigma-Aldrich), and the other chemicals (4-nitrophenyl acetate (N8130:
Sigma-Aldrich), 5,5′-dithiobis(2-nitrobenzoic acid) (D218200:
Sigma-Aldrich), acetylthiocholine iodide (A5771: Sigma-Aldrich), butyrylthiocholine
iodide (B3253: Sigma-Aldrich)) were purchased from local representatives
of well-known commercial companies. On the other hand, the absorbance
rates of each compound were determined by a microplate spectrophotometer
(Multiskan Go, Thermo Scientific).
Çakmak R., Başaran E., Sahin K., Şentürk M, & Durdağı S. (2024). Synthesis of Novel Hydrazide–Hydrazone Compounds and In Vitro and In Silico Investigation of Their Biological Activities against AChE, BChE, and hCA I and II. ACS Omega, 9(18), 20030-20041.
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