- Two-dimensional metal-organic framework nanosheets as a matrix for laser desorption/ionization of small molecules and monitoring enzymatic reactions at high salt concentrations
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Stable 2-D metal-organic framework nanosheets were utilized as a superior clean-background matrix for MALDI-TOF MS analysis of small biomolecules and pollutants in both positive and negative ion modes. The matrix could unusually afford up to 1000 mM of the salt concentrations in the monitoring of the enzymatic hydrolysis of neurotransmitter acetylcholine.
- Liu, Hai-Long,Chang, Yu-Jie,Fan, Ting,Gu, Zhi-Yuan
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- Isoterreulactone A, a novel meroterpenoid with anti-acetylcholinesterase activity produced by Aspergillus terreus
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A new seven-membered lactone type meroterpenoid, isoterreulactone A, was isolated from the solid state fermentation of Aspergillus terreus and its structure was established by various spectral analysis. Isoterreulactone A inhibited acetylcholinesterase with an IC50 value of 2.5 μM while did not inhibit butyrylcholinesterase even at 500 μM. A new seven-membered lactone type meroterpenoid, isoterreulactone A, was isolated from the solid state fermentation of Aspergillus terreus and its structure was established by various spectral analysis. Isoterreulactone A inhibited acetylcholinesterase with an IC50 value of 2.5 μM while did not inhibit butyrylcholinesterase even at 500 μM.
- Yoo, Ick-Dong,Cho, Kyung-Mi,Lee, Chong-Kil,Kim, Won-Gon
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- An improved immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand screening
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Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H]+ m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC50 values were 0.03 ± 0.006 μM and 0.88 ± 0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ± 0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results.
- Vilela, Adriana Ferreira Lopes,Seidl, Cláudia,Lima, Juliana Maria,Cardoso, Carmen Lúcia
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- Proximate Charge Effects. 2. Enthalpies of Solvent Transfer in the Choline-Anhydrocholine Equilibrium
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Equilibrium constants for the choline-anhydrocholine equilibrium, eq 1, +(CH3)3NCH2CH2OH + OH- --> +(CH3)3NCH2CH2O- + H2O, were measured conductometrically in water, aqueous alcohol, and aqueous dimethyl sulfoxide at several temperatures.On going to progressively less H-bonding media, there was a large shift of the equilibrium constant toward anhydrocholine (to the right) accompanied by an even larger exothermic shift of the enthalpy of reaction.Calorimetric measurement of the enthalpies of solvent transfer revealed that, in the case of the aqueous alcohol solvents, the exothermic shift was caused by enhanced solvation of anhydrocholine, +(CH3)3NCH2CH2O-, by the less polar media.In the comparison of aqueous ethanol with aqueous Me2SO, the dominant factor was found to be the very great desolvation of both hydroxide ion and anhydrocholine in the less H-bonding medium.
- Haberfield, Paul,Pessin, Jeffrey
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- Neuro-Regenerative Choline-Functionalized Injectable Graphene Oxide Hydrogel Repairs Focal Brain Injury
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Brain damage is associated with spatial imbalance of cholinergic system, which makes severe impact in recovery of damaged neurons of brain. Therefore, maintenance of cholinergic system is extremely important. Here, we fabricated an injectable hydrogel with acetylcholine-functionalized graphene oxide and poly(acrylic acid). Results revealed that this hydrogel is non-cytotoxic, promotes neurite outgrowth, stabilizes microtubule networks, and enhances the expression of some key neural markers in rat cortical primary neurons. Further, this hydrogel exhibits significant potential in neuro-regeneration and also promotes fast recovery of the sham injured mice brain. Moreover, we found significant enhancement of reactive astrocytes in the hippocampal dentate gyrus region of the sham injured brain, indicating its excellent potential in neural repair of the damaged brain. Finally, above results clearly indicate that this neuro-regenerative hydrogel is highly capable of maintaining the cholinergic balance through local release of acetylcholine in the injured brain, which is crucial for brain repair.
- Pradhan, Krishnangsu,Das, Gaurav,Khan, Juhee,Gupta, Varsha,Barman, Surajit,Adak, Anindyasundar,Ghosh, Surajit
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- Monitoring Enzymatic Reactions in Real Time Using Venturi Easy Ambient Sonic-Spray Ionization Mass Spectrometry
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We developed a technique to monitor spatially confined surface reactions with mass spectrometry under ambient conditions, without the need for voltage or organic solvents. Fused-silica capillaries immersed in an aqueous solution, positioned in close proximity to each other and the functionalized surface, created a laminar flow junction with a resulting reaction volume of 5 pL. The setup was operated with a syringe pump, delivering reagents to the surface through a fused-silica capillary. The other fused-silica capillary was connected to a Venturi easy ambient sonic-spray ionization source, sampling the resulting analytes at a slightly higher flow rate compared to the feeding capillary. The combined effects of the inflow and outflow maintains a chemical microenvironment, where the rate of advective transport overcomes diffusion. We show proof-of-concept where acetylcholinesterase was immobilized on an organosiloxane polymer through electrostatic interactions. The hydrolysis of acetylcholine by acetylcholinesterase into choline was monitored in real-time for a range of acetylcholine concentrations, fused-silica capillary geometries, and operating flow rates. Higher reaction rates and conversion yields were observed with increasing acetylcholine concentrations, as would be expected.
- Jansson, Erik T.,Dulay, Maria T.,Zare, Richard N.
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- UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module
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Despite the prevalence of ortho- and pyrophosphate in biochemistry, operationally simple and versatile high-throughput methodologies for their quantification are lacking. We herein introduce PUB, a module for phosphate detection by continuous UV-spectroscopic monitoring of 5-bromouridine phosphorolysis. The PUB module uses cheaply available, bench-stable reagents and can be employed for continuous and discontinuous reaction monitoring in biochemical assays to detect (pyro-)phosphate concentrations spanning almost 4 orders of magnitude, as demonstrated with representative use cases.
- Eilert, Lea,Schallmey, Anett,Kaspar, Felix
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p. 3432 - 3435
(2022/03/02)
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- Preparation of acetylcholine biosensor for the diagnosis of Alzheimer's disease
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We report herein the design of a novel biosensor sensing strategy for sensitive detection of acetylcholine based on PAMAM-Sal dendrimer. PAMAM-Sal, salicylaldehyde and PAMAM dendrimer have been synthesized by means of condensation. It has been determined that PAMAM-Sal dendrimer was formed by the formation of Schiff base with FT-IR, 1H NMR and UV spectra. In addition, the structure has been supported by elemental analysis. Later, a bienzymatic biosensor system has been developed. The bienzymatic biosensor system with acetylcholine esterase (AChE) and choline oxidase (ChO) was prepared with carbon paste electrode modified with PAMAM-Sal for determination of the amount of acetylcholine. Acetylcholine esterase and choline oxidase enzymes were immobilized onto modified carbon paste electrode by cross-linking with glutaraldehyde. Determination of acetylcholine was carried out by the oxidation of enzymatically produced H2O2 at +0.4 V vs. Ag/AgCl. The linear working range for acetylcholine determination of biosensor was identified. The effects of pH and temperature on the response of the biosensor were examined. Reusability and storage stability of the biosensor were determined. Interference effects of interferants which might be in biologic media on the response of the biosensor were also studied.
- ?olak, ?zlem,Arslan, Fatma,Arslan, Halit,Bodur, Onur Can,Di?li, Ali,Hasano?lu ?zkan, Elvan,Sar?, Nur?en
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- Online Monitoring of Enzymatic Reactions Using Time-Resolved Desorption Electrospray Ionization Mass Spectrometry
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Electrospray ionization mass spectrometry (ESI-MS) is powerful for determining enzymatic reaction kinetics because of its soft ionization nature. However, it is limited to use ESI-favored solvents containing volatile buffers (e.g., ammonium acetate). In addition, lack of a quenching step for online ESI-MS reaction monitoring might introduce inaccuracy, due to the possible acceleration of reaction in the sprayed microdroplets. To overcome these issues, this study presents a new approach for online measuring enzymatic reaction kinetics using desorption electrospray ionization mass spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in a buffered aqueous media (e.g., a solution containing Tris buffer or high concentration of inorganic salts) could be directly detected. Furthermore, by adjusting the pH and solvent composition of the DESI spray, reaction can be online quenched to avoid the postionization reaction event, leading to fast and accurate measurement of kinetic constants. Reaction time control can be obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by acetylcholinesterase. Derived Michaelis-Menten constants Km for these two reactions were determined to be 214 μM and 172 μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine Km and turnover number kcat for trypsin digestion of angiotensin II (Km and kcat are determined to be 6.4 mM and 1.3 s-1, respectively).
- Cheng, Si,Wu, Qiuhua,Xiao, He,Chen, Hao
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p. 2338 - 2344
(2017/08/09)
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- Catalytic supramolecular self-assembled peptide nanostructures for ester hydrolysis
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Essential amino acids in catalytic sites of native enzymes are important in nature inspired catalyst designs. Active sites of enzymes contain the coordinated assembly of multiple amino acids, and catalytic action is generated by the dynamic interactions among multiple residues. However, catalysis studies are limited by the complex and dynamic structure of the enzyme; and it is difficult to exclusively attribute a given function to a specific residue. Minimalistic approaches involving artificial catalytic sites are promising for the investigation of the enzyme function in the absence of non-essential protein components, and self-assembling peptide nanostructures are especially advantageous in this context. Here we demonstrate the design and characterization of an enzyme-mimetic catalytic nanosystem presenting essential residues (Ser, His, Asp). The function of each residue and its combinations on the nanostructures in hydrolysis reaction was studied. The catalytic self-assembled nanostructures were used for efficient ester hydrolysis such as a model substrate (pNPA) and a natural substrate (acetylcholine) highlighting the key role of self-assembly in catalytic domain formation to test the efficiency of the de novo designed catalyst as a catalytic triad model.
- Gulseren, Gulcihan,Khalily, Mohammad Aref,Tekinay, Ayse B.,Guler, Mustafa O.
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supporting information
p. 4605 - 4611
(2016/07/11)
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- Bioactive MIL-88A Framework Hollow Spheres via Interfacial Reaction In-Droplet Microfluidics for Enzyme and Nanoparticle Encapsulation
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Functional bio-MOF hollow spheres with controlled size in the 35-2000 μm range were successfully synthesized by interfacial reaction using a continuous-flow droplet microfluidic system in a single step and one-flow strategy. The architecture of MIL-88A frameworks was extended from single-shell to double-shell hollow spheres. Moreover, various functional nanoparticles (silica, cobalt, and UiO-66(Zr) MOF) were directly encapsulated in the single-shell hollow spheres, while maintaining the functionality of the cargo. In particular, three kinds of enzymes (glycerol dehydrogenase, horseradish peroxidase, and acetylcholinesterase) were also encapsulated inside the single-shell hollow spheres under mild conditions. The catalytic activity and the superior recyclability of the encapsulated enzymes were demonstrated against free enzymes.
- Jeong, Guan-Young,Ricco, Raffaele,Liang, Kang,Ludwig, Johanna,Kim, Jin-Oh,Falcaro, Paolo,Kim, Dong-Pyo
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p. 7903 - 7909
(2015/12/20)
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- Programmed enzyme-mimic hydrolysis of a choline carbonate by a metal-free 2-aminobenzimidazole-based cavitand
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The hydrolysis of a choline carbonate through a metal-free, enzyme-like mechanism has been achieved using a 2-aminobenzimidazole-based deep cavitand as catalyst. The supramolecular catalysis involves three steps: host-guest binding, carbamoylation and enzyme-like hydrolysis. Interestingly the rate-determining step proceeds through a programmed hydrolysis of carbamoylcholine-cavitand intermediate that may be driven by water molecules surrounding the benzimidazole walls of the cavity.
- Soberats, Bartolome,Sanna, Elena,Martorell, Gabriel,Rotger, Carmen,Costa, Antoni
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supporting information
p. 840 - 843
(2014/03/21)
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- Photoluminescent C-dots@RGO probe for sensitive and selective detection of acetylcholine
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We have developed a sensitive and selective photoluminescence (PL) quenching assay for the detection of acetylcholine (ACh) that uses reduced graphene oxide decorated with carbon dots (C-dots@RGO). The highly stable C-dots@RGO synthesized from catechin and graphene oxide through a hydrothermal reaction displays excitation-wavelength dependence of PL. Acetylcholinesterase (AChE) converts ACh to choline, which in turn is oxidized by choline oxidase (ChOx) to produce betaine and H2O2 that generates the reactive oxygen species (ROS). The as-produced ROS induces PL quenching of the C-dots@RGO through an etching process. With respect to sensitivity, the optimal reaction/sensing temperature and pH are 37 C and 9.0, respectively, using C-dots@RGO (0.4 mg·mL-1) and AChE and ChOx at the activities of 0.5 and 0.1 unit·mL-1, respectively. The PL intensity (excitation/emission wavelengths 365/440 nm) of the C-dots@RGO is inversely proportional to the concentration of ACh over a range of 0.05-10 nM (r = 0.997), with a limit of detection (signal-to-noise ratio 3) of 30 pM. We have validated this assay by determination of concentrations of ACh in plasma and blood samples, with results of 2.6 ± 0.8 nM (n = 5) and 6.8 ± 0.4 nM (n = 5), respectively. Our study opens an avenue for the detection of various analytes by use of C-dots@RGO in conjunction with different enzymes, substrates, and/or inhibitors.
- Wang, Chen-I,Periasamy, Arun Prakash,Chang, Huan-Tsung
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p. 3263 - 3270
(2013/06/05)
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- Real-time fluorometric assay for acetylcholinesterase activity and inhibitor screening through the pyrene probe monomer-excimer transition
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A choline labeled pyrene probe (Py-Ch) was designed and synthesized. Poly(vinylsulfonate) (PVS) could induce Py-Ch aggregation. The aggregation and deaggregation process could be finely controlled by the acetylcholinesterase (AChE) enzymatic hydrolysis of Py-Ch. The resulting excimer-monomer transition provided a facile way for real-time AChE activity fluorometric assay and inhibitor screening.
- Chen, Jian,Liao, Dongli,Wang, Yan,Zhou, Huipeng,Li, Wenying,Yu, Cong
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supporting information
p. 2132 - 2135
(2013/06/26)
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- Zwitterionic and anionic multinuclear pentacoordinate silicon(IV) complexes with bridging (R,R)-tartrato(4-) ligands and SiO5 skeletons: Synthesis and reactivity in aqueous solution
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Two nutrients in one molecule: A zwitterionic λ5Si, λ5Si′-disilicate (1) was synthesized and characterized. It contains ligands that exclusively derive from natural products ((R,R)-tartaric acid, choline). Hydrolysis of 1 yields 2, which shows a remarkable kinetic stability in water. Upon dissolution of 1 and 2 in water, the nutrients choline and orthosilicic acid are formed by hydrolysis Copyright
- Theis, Bastian,Weiss, Joerg,Lippert, W. Peter,Bertermann, Ruediger,Burschka, Christian,Tacke, Reinhold
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p. 2202 - 2206
(2012/04/10)
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- Predominant role of basicity of leaving group in α-effect for nucleophilic ester cleavage
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It has been found that α-effects in nucleophilic reactions, unexpectedly large nucleophilicity due to adjacent unpaired electrons, are strongly dependent on the structure of substrate. The nucleophilic cleavages of 4-nitrobenzoate esters and 4-methylbenzo
- Nomura, Yasuo,Kubozono, Takayasu,Hidaka, Makoto,Horibe, Mineko,Mizushima, Naoki,Yamamoto, Nobuyuki,Takahashi, Toshio,Komiyama, Makoto
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- Interactions of chiral quinuclidin-3-yl benzoates with butyrylcholinesterase: Kinetic study and docking simulations
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Both enantiomers of quinuclidin-3-yl benzoate (RQBz and SQBz) were synthesized in order to examine the stereoselectivity of the hydrolysis of these esters catalyzed by horse serum butyrylcholinesterase (BChE). The hydrolysis of benzoylcholine (BzCh) was a
- Primozic, Ines,Hrenar, Tomica,Tomic, Srdanka,Meic, Zlatko
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p. 608 - 614
(2007/10/03)
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- Determination of butyrylcholinesterase inhibition using ion transfer across the interface between two immiscible liquids
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An investigation was made into the inhibition of the enzyme butyrylcholinesterase by paraoxon (diethyl p-nitrophenyl phosphate), using butyrylcholine chloride as the substrate. Experimental measurement was based on the transfer of the butyrylcholine cation across the interface between water and 1,2-dichloroethane using cyclic voltammetry. By this method it was possible to determine the rate constants for both the inhibition of the enzyme and the hydrolysis of butyrylcholine.
- Beattie,Infelta,Girault
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- A FACILE ONE-STEP SYNTHESIS OF 5'-PHOSPHATIDYLNUCLEOSIDES BY AN ENZYMATIC TWO-PHASE REACTION.
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Phospholipase D from Streptomyces effectively catalyzed the transfer reaction of the phosphatidyl residue from phosphatidylcholines to the 5'-hydroxyl group of nucleosides in a two-phase system.A variety of 5'-phosphatidylnucleosides were easily prepared in high yields by this reaction.
- Shuto, Satoshi,Ueda, Shigeru,Imamura, Shigeyuki,Fukukawa, Kiyofumi,Matsuda, Akira,Ueda, Tohru
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p. 199 - 202
(2007/10/02)
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