P-nitrophenyl Phosphate Research Articles

Novel dual-emission sulfur quantum dot sensing platform for quantitative monitoring of pesticide 2,4-dichlorophenoxyacetic acid

In this work, a novel environment-friendly dual-emission Rhodamine B modified sulfur quantum dots (RhB-SQDs) sensing platform was established to economically monitor organochlorine pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) through regulating the activity of alkaline phosphatase (ALP). This dual emission RhB-SQDs exhibited excellent fluorescence and high photostability with emission wavelengths of 455 nm and 580 nm. ALP catalyzed the hydrolysis of the substrate p-nitrophenyl phosphate to p-nitrophenol, which quenched RhB-SQDs fluorescence at 455 nm due to the internal filtration effect, but had no effect the fluorescence intensity of RhB-SQDs at 580 nm. When 2,4-D was present, the activity of ALP was specifically inhibited and enzymatic reaction was interrupted, leading to the reduction of p-nitrophenol production, so the fluorescence of RhB-SQDs at 455 nm was restored. It demonstrated a good linear relationship between the concentration of 2,4-D and F455/F580 in the range of 0.050–0.500 μg mL−1, with a detection limit of 17.3 ng mL−1. The dual-emission fluorescent probe was successfully realized in the identification of 2,4-D in natural water samples and vegetables with the advantages of exceptional accuracy, immunity to interference, and selectivity. The platform offers a fresh look at pesticide monitoring and has the potential to prevent pesticide-related health issues.

Bifunctional reduced graphene oxide/polyelectrolyte/NiFe layered double hydroxide composites for efficient catalyzed dephosphorylation and 4-nitrophenol reduction

Dephosphorylation that separates phosphate groups from substrates is a significant reaction for both living organisms and environmental protection. Meanwhile, 4-nitrophenol (4-NP) is known to be a popular organic compound used for pesticides production, which is genotoxic and carcinogenic to humans and animals. Herein, we have developed a bifunctional catalyst that can be used for simultaneous dephosphorylation and 4-nitrophenol (4-NP) reduction when para-nitrophenyl phosphate (p-NPP) serves as the model compound. This bifunctional catalyst was obtained by in-situ growth of NiFe layered double hydroxide on the reduced graphene oxide/polyelectrolytes assembly nanolayers (denoted as rGP3.5/NiFe LDH). The results demonstrate that the rGP3.5/NiFe LDH can activate dephosphorylation of p-NPP at 20 °C and the catalytic efficiency increases significantly with increasing temperature in the range of 20 °C to 90 °C. Moreover, by-product 4-NP formed through p-NPP dephosphorylation is further fully converted to 4-aminophenol by the rGP3.5/NiFe LDH within 15 min. As far as we know, this is the first example for both dephosphorylation and 4-NP reduction through NiFe LDH as bifunctional catalysts, which paves the way for the development of multiple potential applications for NiFe LDH.

Bioactive Compounds from Mimosa pudica Leaves Extract with Their α- glucosidase and Protein Tyrosine Phosphatase 1B Inhibitory Activities in vitro and in silico Approaches

<p>Background: Mimosa pudica Linn has been used in traditional medicine to support the treatment of type 2 diabetes. In the present study, we aimed to isolate and evaluate α-glucosidase and Protein Tyrosine Phosphatase 1B (PTP1B) inhibitory activities of bioactive compounds from Mimosa pudica’s leaf extract. <p> Methods: Mimosa pudica leaves were extracted with 80% of ethanol. Bioactive compounds were isolated using a column chromatographic technique and elucidated the structure based on the nuclear magnetic resonance and electrospray ionization mass spectrometry spectral data. The α- glucosidase and PTP1B inhibitory activities of the isolated compounds were evaluated using pnitrophenyl phosphate and p-nitrophenyl-α-D-glucopyranoside as a substrate, respectively. Molecular docking and molecular dynamics are used to study the interaction between isolated compounds and proteins. Lipinski’s rule of five was used to evaluate the drug-like properties of isolated compounds. Predict pharmacokinetic parameters were evaluated using the pkCSM tool. <p> Results: Protocatechuic acid and syringic acid were isolated and identified using spectroscopic methods. Protocatechuic acid and syringic acid considerably inhibited α-glucosidase enzyme at IC<sub>50</sub> values of 416.17 ± 9.41 μM and 490.78 ± 9.28 μM, respectively. Furthermore, protocatechuic acid and syringic acid expressed strong PTP1B inhibitory activity at IC<sub>50</sub> values of 248.83 ± 7.66 μM and 450.31 ± 7.77 μM, respectively. Molecular docking and molecular dynamics results showed the interactions of protocatechuic acid and syringic acid with amino acids of PTP1B and α-glucosidase enzyme. Lipinski’s rule of five and absorption, distribution, metabolism, excretion, and toxicity studies predicted that protocatechuic acid and syringic acid have drug-likeness properties. In molecular docking simulation, protocatechuic acid and syringic acid gave relatively negative free binding energies and interacted with many amino acids in the active sites of PTP1B and α-glucosidase. The molecular dynamics simulation results of the complexes were also relatively stable. <p> Conclusion: Our results showed that protocatechuic and syringic acids could be promising compounds for type 2 diabetes treatment.</p>

Spontaneously super-hygroscopic MOF-gel microreactors for efficient detoxification of nerve agent simulant in atmospheric environments

We design a spontaneously super-hygroscopic alkaline microreactor by photoinduced integration of Zr-MOF catalyst and alkaline poly(dimethylaminoethyl acrylate) onto LiCl-salinized poly(N-isopropylacrylamide) gel, generating a microenvironment with non-volatile polymeric base and non-bulk water for detoxification of a typical nerve agent simulant, dimethyl 4-nitrophenyl phosphate (DMNP), in atmospheric environments. The super-hygroscopic gel with directional microchannels facilitates the absorption, transport and storage of water under different humidities, and exhibits fast hygroscopic efficiency with a high moisture absorption capacity of 6.4 g g−1. The integrated microreactor achieves simultaneous water uptake and catalytic hydrolysis of DMNP in an atmospheric environment with an initial half-life of ∼1.9 h and a final conversion of 95.5%, which is attributed to the synergistic catalytic microenvironment of the exposed Zr-MOF catalyst, the polymeric base, and the water from moist air. This work provides a feasible strategy for designing spontaneously hygroscopic microreactors for detoxifying nerve agent simulants in atmospheric environments.

Isolation and characterization of the first phosphodiesterase (Bj-PDE) from the venom of Bothrops jararacussu snake

Phosphodiesterases are exonucleases that sequentially hydrolyse phosphodiester bonds of polynucleotides from the 3′-end and release 5-mononucleotides. After more than one decade without any advance in the study of Bothropic phosphodiesterases, we described here the isolation of the first phosphodiesterase from Bothrops jararacussu, which we named Bj-PDE. A five-step column chromatography procedure (size exclusion, hydrophobic interaction, cation exchange, lentil lectin affinity, and blue sepharose affinity) enabled isolation of Bj-PDE with preserved and stable enzymatic activity (bis(p-nitrophenyl) phosphate substrate), Km = 6.9 mM (± 0.7 mM), kcat/Km = 1.7 × 104 M−1 s−1 (± 0.2 × 104 M−1 s−1), MW = 116 kDa (SDS-PAGE), optimum activity around 45 °C at pH 8.0, and stability for 81 days at different storage temperatures (8, −20, and − 80 °C). Ca2+ and Mg2+ ions positively influenced Bj-PDE activity, while EDTA had the opposite action. Zn2+ restored >50 % of enzyme activity after its inhibition by EDTA. The Bj-PDE partial sequence identified by mass spectrometry was very similar to the sequence of BATXPDE1 from Bothrops atrox, which was evolutionarily close to this new PDE. Therefore, our study represents an important progress on the isolation of this minor toxin and sheds new lights on the properties and bioprospection of bothropic phosphodiesterases.

A new fluorescent biosensor based on inner filter effect and competitive coordination with the europium ion of non-luminescent Eu-MOF nanosheets for the determination of alkaline phosphatase activity in human serum

The abnormal level of alkaline phosphatase (ALP) in human blood stream indicates health problem. A novel fluorescent sensing system based on competitive coordination with europium ions (Eu3+) integrated in a non-luminescent lanthanide-metal organic framework nanosheets (NO2-Eu-MOF NS) was developed for ALP analysis in human serum. The fluorescence signal is turned-on by the antenna ligand (1,4-phenylenediphosphonic acid (PEPP)) that competitively coordinates with Eu3+ ions from the nanosheet to produce emissive species (Eux(PEPP)y(NO2BDC)z) in-situ. 4-Nitrophenyl phosphate (PNPP) is a substrate of ALP and specifically hydrolyzed into 4-nitrophenol. PNPP exhibits a strong absorption in the UV-Vis region (250–400 nm), which covers the excitation wavelength (280 nm) of Eux(PEPP)y(NO2BDC)z. Therefore, the existing of PNPP causes Eux(PEPP)y(NO2BDC)z non-emissive and, after hydrolysis with ALP, Eux(PEPP)y(NO2BDC)z can be excited and give a detectable fluorescence signal. Based on the fluorescence change, quantitative analysis of ALP activity is thus achievable. This biosensing system was demonstrated in the determination of ALP activity with good linear relationship in the range of 5–1000 U L−1. The limit of detection was 1.1 U L−1 (S/N = 3). The result reveals that the biosensor exhibits excellent sensing performance and anti-interference ability. It could be potentially utilized for clinical routine analysis of ALP activity in human serum.

Dual-Function Detoxifying Nanofabrics against Nerve Agent and Blistering Agent Simulants

The development of functional materials that can detoxify multiple chemical warfare agents (CWAs) at the same time is of great significance to cope with the uncertainty of CWA use in real-world situations. Although many catalysts capable of detoxifying CWAs have been reported, there is still a lack of effective means to integrate these catalytic-active materials on practical fibers/fabrics to achieve effective protection against coexistence of a variety of CWAs. In this work, by a combination of electrospinning and in situ solvothermal reaction, PAN@Zr(OH)4@MOF-808 nanofiber membranes were prepared for detoxification of both nerve agent and blistering agent simulants dimethyl 4-nitrophenyl phosphate (DMNP) and 2-chloroethyl ethyl sulfide (CEES). Under the catalytic effect of the MOF-808 component, DMNP hydrolysis with a half-life as short as 1.19 min was achieved. Meanwhile, an 89.3% CEES removal rate was obtained within 12 h by adsorption and catalysis of MOF-808 and Zr(OH)4 components at ambient conditions, respectively. PAN@Zr(OH)4@MOF-808 nanofiber membranes also showed a superior blocking effect on CEES compared to bare PAN and PAN@Zr(OH)4 nanofiber membranes. Simultaneous protection against DMNP and CEES showed effective inhibition of both simulants for at least 2 h. The preparation method also imparted intrinsically good interfacial adhesion between the components, contributing to the excellent recycling stability of PAN@Zr(OH)4@MOF-808 nanofiber membranes. Therefore, the prepared composite nanofabrics have great application potential, which provides a new idea for the construction of broad-spectrum protective detoxification materials.

A novel and sensitive ratiometric fluorescent quantum dot-based biosensor for alkaline phosphatase detection in biological samples via the inner-filter effect.

Alkaline phosphatase (ALP) is an important biomarker whose abnormal level in activity is associated with hepatobiliary, skeletal, and renal diseases as well as cancer. Herein, we synthesized ZnSe@ZnS quantum dots (ZnSe@ZnS QDs) and Mn-doped ZnS quantum dots (Mn:ZnS QDs) as fluorophores to establish the ratiometric fluorescent assay for ALP activity detection in biological samples. p-Nitrophenyl phosphate (PNPP) was used as a substrate for ALP, and the overlaps between absorption spectra of PNPP and excitation spectra of QDs resulted in sharp fluorescence quenching. Under the catalysis of ALP, PNPP was hydrolyzed into p-nitrophenol (PNP), which caused a red shift of absorption band of PNPP and fluorescence recovery of Mn:ZnS QDs (585 nm). However, the overlaps between absorption spectra of PNP and emission spectra of ZnSe@ZnS QDs led a further quenching of ZnSe@ZnS QDs (405 nm). Therefore, the ratiometric fluorescent signals (F 585/F 405) were associated with activity of ALP based on bidirectional responses of QDs to the concentration of PNPP. Under the optimum conditions, the method exhibited a good linear relationship from 4 to 96 U per L (R 2 = 0.9969) with the detection limit of 0.57 U per L. Moreover, the method was successfully applied for detecting the ALP activity in a complex biological matrix (human serum and HepG2 cells) with impressive specificity. In particular, the complicated chemical modifications of QDs and pretreatments of biological samples were not required in the whole detection procedures. Therefore, it not only provided a sensitive, specific and simple approach to clinical ALP activity detection, but it also provided support for early diagnosis of diseases.

Steady-state kinetic analysis of reversible enzyme inhibitors: A case study on calf intestine alkaline phosphatase.

Enzymes are important drug targets and inhibition of enzymatic activity is an important therapeutic strategy. Enzyme assays measuring catalytic activity are utilized in both the discovery and development of new drugs. Colorimetric assays based on the release of 4-nitrophenol from substrates are commonly used. 4-Nitrophenol is only partly ionized to 4-nitrophenolate under typical assay conditions (pH 7-9) leading to under-estimation of product formation rates due to the much lower extinction coefficient of 4-nitrophenol compared to 4-nitrophenolate. Determination of 4-nitrophenol pKa values based on absorbance at 405nm as a function of experimental pH values is reported, allowing for calculation of a corrected extinction coefficient at the assay pH. Characterization of inhibitor properties using steady-state enzyme kinetics is demonstrated using calf intestine alkaline phosphatase and 4-nitrophenyl phosphate as substrate at pH ∼8.2. The following kinetic parameters were determined: Km=40±3µM; Vmax=72.8±1.2µmolmin-1mg protein-1; kcat=9.70±0.16s-1; kcat/Km=2.44±0.16 × 105 M-1s-1 (mean±SEM, N=4). Sodium orthovanadate and EDTA were used as model inhibitors and the following pIC50 values were measured using dose-response curves: 6.61±0.08 and 3.07±0.03 (mean±SEM, N=4). Rapid dilution experiments determined that inhibition was reversible for sodium orthovanadate and irreversible for EDTA. A Ki value for orthovanadate of 51±8nM (mean±SEM, N=3) was determined. Finally, data analysis and statistical design of experiments are discussed.

Real Time Paper Based Detection of Streptococcus bovis using Chromogenic Substrate in Resource Constrained Environments

Healthcare management faces a variety of difficulties in Circumstances with limited resources. Accurate diagnosis is the first step in disease prevention and therapy that works However, the diagnostic tools that are available in the economically advanced world are frequently of little utility in underdeveloped nations including Pakistan. The gram-positive bacterium Streptococcus bovis is an opportunistic pathogen that can cause various infections from superficial skin infections to severe and potentially fatal invasive diseases. Objective: To design a platform in environments with limited resources for the quick detection of Streptococcus bovis. Methods: A paper based analytical device (PAD) has been created with enrichment in sterile PYP broth for 1.5 hours and used for the detection of alkaline phosphatase activity using the chromogenic substrate Para-Nitrophenyl Phosphate (PNPP), the specie could be found in clinical samples. A coagulase test and sample Gram staining was conducted with the test. Qualitative detection was evaluated by visual detection while quantitative analysis is carried out using Image J software. Results: Alkaline Phosphatase ( S. bovis) reacts with the PNPP substrate (5.7 mM) in the presence of PAD. Concentrations below 4.5x104 cfu mL resulted in the detection of a color change. The micro PADs were incubated at 37 °C for 3–4 hours before reaction. Colored product (yellow) indicated the presence of S.bovis. Conclusion: Within 2 hours including enrichment time the test may identify Streptococcus bovis up to 104 CFUmL-1

ZnFe2O4/ZrO2/NaX zeolite nanocomposite catalyst: elaboration and its application for the removal of dimethyl 4-nitrophenyl phosphate (DMNP) chemical nerve agent simulant from water solution

ZnFe2O4/ZrO2/NaX zeolite nanocomposite catalyst: elaboration and its application for the removal of dimethyl 4-nitrophenyl phosphate (DMNP) chemical nerve agent simulant from water solution

Kinetic and thermodynamic studies of novel acid phosphatase isolated and purified from Carthamus oxyacantha seedlings

Acid phosphatase (ACP) is a key enzyme in the regulation of phosphate feeding in plants. In this study, a new ACP from C. oxyacantha was isolated to homogeneity and biochemically described for the first time. Specific activity (283 nkat/mg) was found after 2573 times purification fold and (17 %) yield. Using SDS-PAGE under denaturing and nondenaturing conditions, ACP was isolated as a monomer with a molecular weight of 36 kDa. LC-MS/MS confirmed the presence of this band, suggesting that C. oxycantha ACP is a monomer. The enzyme could also hydrolyze orthophosphate monoester with an optimal pH of 5.0 and a temperature of 50 °C. Thermodynamic parameters were also determined (Ea, ΔH°, ΔG°, and ΔS°). ACP activity was further studied in the presence of cysteine, DTT, SDS, EDTA, β-ME, Triton-X-100 H2O2, and PMSF. The enzyme had a Km of 0.167 mM and an Ea of 9 kcal/mol for p-nitrophenyl phosphate. The biochemical properties of the C. oxyacantha enzyme distinguish it from other plant acid phosphatases and give a basic understanding of ACP in C. oxyacantha. The results of this investigation also advance our knowledge about the biochemical significance of ACP in C. oxyacantha. Thermal stability over a wide pH and temperature range make it more suitable for use in harsh industrial environments. However, further structural and physiological studies are anticipated to completely comprehend its important aspects in oxyacantha species.

Rational Design of a Zr-MOF@Curli-Polyelectrolyte Hybrid Membrane toward Efficient Chemical Protection, Moisture Permeation, and Catalytic Detoxification.

Developing high-performance protective materials is important for soldiers and civilians who are exposed to the atmosphere of highly toxic chemical warfare agents (CWAs). Polyelectrolyte membranes are promising candidates with excellent chemical resistance and moisture permeability, but they cannot efficiently degrade CWAs. Here, we design and prepare a hybrid membrane through in situ growth of catalytically active zirconium-based metal-organic frameworks (Zr-MOFs) on a polyelectrolyte membrane mediated by biofilm-inspired curli nanofibers (CNFs). Superior to the bare polyelectrolyte membrane, the prepared MOF-808@CNF-PQ hybrid membrane exhibits improved rejection of the nerve agent simulant dimethyl methyl phosphonate (DMMP) vapor and permeation of the water vapor by 113 and 45%, respectively. The water/DMMP selectivity of the hybrid membrane reaches 498.6, approximately 13 times that of the commercial polyelectrolyte membrane Nafion 117. In addition, the hybrid membrane possesses appreciable catalytic activity for the hydrolysis of the nerve agent simulant dimethyl 4-nitrophenyl phosphate (DMNP) with a half-life of ∼38 min. Nanomechanical characterization results based on atomic force microscopy (AFM) techniques demonstrate the critical role of CNFs in mediating Zr-MOF nucleation and the dominant effect of electrostatic interactions on self-assembly of CNFs on polyelectrolyte base. It is also confirmed that the Zr-MOF toppings serve as the key components in physically adsorbing and chemically degrading the DMNP molecules through multiple strong intermolecular interactions. Our work offers a rational strategy to develop advanced membranes toward efficient chemical protection, moisture permeation, and catalytic detoxification against CWAs.

Elevated serum alkaline phosphatase correlates with postoperative cognitive dysfunction: A retrospective cohort study based on STROBE statement.

Little is known about the association between serum alkaline phosphatase (ALP) levels and postoperative cognitive dysfunction (POCD) after general anesthesia. Thus, we investigated the association of serum ALP levels with POCD in patients who underwent surgery with general anesthesia in a retrospective cohort study. We retrospectively collected data from patients who underwent surgery with general anesthesia between May 2016 and June 2020. Serum ALP activity was detected using a p-nitrophenyl phosphate assay. Pre-and postoperative cognitive function were evaluated using the Chinese version of the Mini-Mental State Examination. Univariate and multivariate logistic regression were used to explore the effect of ALP on cognitive function. The incidence of POCD was 13.5%. Compared with the control group, the POCD group had higher ALP levels. The neuropsychological test results suggested that the scores of most items were lower in the POCD group than in the non-POCD group. Univariate logistic regression indicated that increased ALP levels were significantly associated with cognitive dysfunction (odds ratio = 1.15, 95% confidence interval: 1.13-1.18, P = .000). Multivariate regression showed that elevated ALP was still associated with POCD after adjusting for confounding factors (odds ratio = 1.16, 95% confidence interval: 1.13-1.18, P = .000). The spline regression model indicated the dose-response associations between ALP level and POCD risk (P for nonlinear trend < .001). Our study indicated that elevated serum ALP was an independent predictive factor of POCD at the 3-month follow-up. The occurrence of POCD could be associated with inflammatory status.

Construction of MOF-loaded polypropylene nonwoven fabrics for fast catalytic hydrolysis of chemical warfare agent simulants

• MOF-808/fiber composite was fabricated using tannic acid (TA) hybrid coating as the mediating layer. • The as-prepared MOF-808/fiber composite with 23.4 % MOF-808 displayed a high surface area (340 m 2 /g). • The obtained PP fabrics displayed a half-life of 1.3 min for dimethyl p -nitrophenyl phosphate (DMNP) degradation. • The PP@TA-APTES@MOF-808 catalyst has good stability for DMNP detoxification . Zirconium-based metal-organic frameworks (Zr-MOFs) have emerged as promising candidates for the catalyzed hydrolysis of nerve agents, but facile methods for their integration into textiles are still lacking. Herein, we report a strategy for the preparation of MOF-808-loaded polypropylene (PP) nonwoven fabrics mediated by a tannic acid (TA) and 3-aminopropyltriethoxysilane (APTES) hybrid coating (TA-APTES). The as-prepared PP nonwoven fabric displayed a rapid catalytic degradation rate for dimethyl p -nitrophenyl phosphate (DMNP) with a half-life of 1.3 min in a basic buffered condition.

Ultrastructural Changes in the Kidneys and Biochemical Parameters of Blood and Urine in Rats under Acute Intoxication with O,O-Diethyl O-(4-nitrophenyl) Phosphate

Ultrastructural Changes in the Kidneys and Biochemical Parameters of Blood and Urine in Rats under Acute Intoxication with O,O-Diethyl O-(4-nitrophenyl) Phosphate

Chemo-enzymatic synthesis of 5-substituted ribavirin analogs: Unexpected cooperative effect in the interaction of 5-alkyloxymethyl 1,2,4-triazol-3-carboxamides with E. coli purine nucleoside phosphorylase active site

A series of 5-alkyl/aryl-oxymethyl-ribavirin analogs was synthesized. Surprisingly, ability of E. coli purine nucleoside phosphorylase (PNP) to synthesize ribonucleosides and 2′-deoxyribonucleosides having structurally diverse alkyl(aryl)oxymethyl substituents at the 5 position of 1,2,4-triazol-3-carboxamide has been discovered. It has been shown that the enzymatic synthesis of 2′-deoxyribonucleosides proceeds with better conversion and is faster (up to 50 h) than the synthesis of ribonucleosides (more than 300 h). Therefore, ribonucleosides were synthesized by chemical glycosylation of base triazoles by 2,3,4,5-tetra-O-acetyl-D-ribose in the presence of bis(4-nitrophenyl) phosphate. 2′-Deoxyribonucleosides were synthesized by the transglycosylation reaction with E. coli PNP. For the first time, based on the kinetic parameters of the 2′-deoxyribonucleosides synthesis process, the positive cooperativity effect of wild type PNP active sites was discovered. The antiviral activity of nucleosides has been tested against herpes simplex virus type 1 (HSV-1) and influenza virus H5N1. The cytotoxicity and antiviral activity of the synthesized nucleosides differ, and it is impossible to draw an unambiguous conclusion about the structure-activity correlation.

Effects of p-Hydroxybenzaldehyde, Vanillin, and Syringaldehyde on Protein Tyrosine Phosphatase 1B Activity

Background: The PTP 1B is a negative regulator of insulin signal transduction and hence, serves as a therapeutic target in the treatment of diabetes. Objective: The present study investigated the inhibitory effects of p-hydroxybenzaldehyde, vanillin, and syringaldehyde on the activity of protein tyrosine phosphatases phosphatase 1B (PTP 1B) in vitro. Method: The PTP 1B inhibitory assay and mode of inhibition of the three compounds were determined using p-nitrophenyl phosphate (p-NPP) in a 96 well microplate. Molecular docking was used to predict the binding affinities of the compounds with the PTP 1B. Results: The results showed that syringaldehyde exhibited significantly (p&lt; 0.05) higher PTP 1B inhibitory activity (IC50: 12.75 µM) compared to p-hydroxybenzaldehyde (IC50: 33.79 µM) and vanillin (IC50: 42.82 µM) as well as the standards suramin (IC50: 28.35 µM) and ursolic acid (IC50: 19.45 µM). Syringaldehyde and vanillin showed uncompetitive inhibition whereas, p-hydroxybenzaldehyde showed a mixed inhibition type. The molecular docking simulation predicted negative binding energies of -5.0 kcal/mol, -5.5 kcal/mol, and -5.5 kcal/mol for p-hydroxybenzaldehyde, vanillin, and syringaldehyde respectively. Conclusion: Syringaldehyde showed higher inhibition of PTP 1B compared to other phenolic aldehydes and could be the mechanism of its antidiabetic activity. Hence, further studies are warranted to confirm the efficacy and toxicity of the compound

Zymography assisted quick purification, characterization and inhibition analysis of K. pneumoniae alkaline phosphatase by mercury and thiohydroxyal compounds

In-gel hydrolysis of para-nitrophenyl phosphate (p-NPP) to yellow colored para-nitrophenol was used to locate precisely the K. pneumoniae alkaline phosphatase (Kp-ALKP) on 7% native PAGE. Subsequent removal of the yellow-stained band and electroelution yielded a 54 kDa, Kp-ALKP with Km, Vmax and kcat values of (0.7 ± 0.02) mM, (80 ± 4.5) μmol min−1 and (39.2 ± 2.2) × 104 s−1 respectively for p-NPP. Kp-ALKP was optimally active at 70 °C and pH 7.2 that was activated by Mg2+, Ca2+, Co2+ and inhibited by EDTA, PO4, Pb2+, Cu2+ and Hg2+. The enzyme was trypsin resistant and retained 75% activity in presence of 10 mM PO4 and 65% activity at 3 mM Hg2+ showing it's PO43− irrepressibility and Hg2+-tolerance. Molecular dynamics simulation revealed increased structural stability of Kp-ALKP at 70 °C that accounts for it's optimal temperature. Zymography revealed that both DTT and β-mercaptoethanol induced activity loss accompanied by mobility retardation of Kp-ALKP on 7% native PAGE. These results and in Silico analysis shows that both DTT and βME reduce the C308–C358 disulfide bond, leading to an open conformation of the enzyme. However, Hg2+ had negligible effect on the in-gel mobility of Kp-ALKP indicating it's plausible non-covalent interaction with surface-accessible amino-acids without significant conformational change. For the first time our study reveals the zymography as an easy, inexpensive and convenient tool for quick purification, characterization and conformational analysis of K. pneumoniae alkaline phosphatase.

Tetranuclear iron(III) complexes with a carboxylate-rich ligand as synthetic mimics of phosphoesterases in aqueous media

The characterization and catalytic activities of two tetranuclear Fe(III) complexes of a carboxylate-rich ligand, N,N’-Bis[2-carboxybenzomethyl]-N,N’ -Bis[carboxymethyl]-1,3-diaminopropan-2-ol (H5ccdp) ligand towards the hydrolysis of p-nitrophenyl phosphate (PNPP) and bis(p-nitrophenyl) phosphate (BNPP) substrates in aqueous systems are described. Kinetic investigations were carried out using UV–vis spectrophotometric techniques at 25 °C and 37 °C and varied pH conditions (7 – 9.5). The kinetic studies showed that the turnover rate (kcat) values for the hydrolysis of PNPP by [Fe4(ccdp)2(μ-O)(μ-OH)(d-GluA)2]3- were marginally higher than [Fe4(ccdp)2(OH)2(o-phth)2]4- at 9.24 × 10-6 s−1 (pH 8 and 37 °C) and 4.27 × 10-7 s−1 (pH 8 and 25 °C), respectively. However, similar comparisons among the BNPP hydrolysis showed the turnover rate values for [Fe4(ccdp)2(OH)2(o-phth)2]4- was marginally better than [Fe4(ccdp)2(μ -O)(μ –OH)(d-GluA)2]3- with values of 1.85 × 10-6 s−1 (pH 8 and 37 °C) and 6.23 × 10-7 (pH 8 and 37 °C). ESI-MS techniques detected catalyst-substrate adduct species for both metal complexes with PNPP. Additionally, both complexes also showed evidence for complex-substrate-nucleophile adduct formation with both BNPP and PNPP substrates via ESI-MS. Based on the kinetic activity and structural information obtained in this study, a reaction mechanism for the hydrolysis of phosphorester has been proposed.