62-49-7Relevant articles and documents
Two-dimensional metal-organic framework nanosheets as a matrix for laser desorption/ionization of small molecules and monitoring enzymatic reactions at high salt concentrations
Liu, Hai-Long,Chang, Yu-Jie,Fan, Ting,Gu, Zhi-Yuan
, p. 12984 - 12987 (2016)
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.
An improved immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand screening
Vilela, Adriana Ferreira Lopes,Seidl, Cláudia,Lima, Juliana Maria,Cardoso, Carmen Lúcia
, p. 53 - 57 (2018)
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.
Neuro-Regenerative Choline-Functionalized Injectable Graphene Oxide Hydrogel Repairs Focal Brain Injury
Pradhan, Krishnangsu,Das, Gaurav,Khan, Juhee,Gupta, Varsha,Barman, Surajit,Adak, Anindyasundar,Ghosh, Surajit
, p. 1535 - 1543 (2019)
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.
UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module
Eilert, Lea,Schallmey, Anett,Kaspar, Felix
, p. 3432 - 3435 (2022/03/02)
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.
Online Monitoring of Enzymatic Reactions Using Time-Resolved Desorption Electrospray Ionization Mass Spectrometry
Cheng, Si,Wu, Qiuhua,Xiao, He,Chen, Hao
, p. 2338 - 2344 (2017/08/09)
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).