1.Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae.
Alizadeh A1, Talebi-Jahromi K2, Hosseininaveh V3, Ghadamyari M4. J Insect Sci. 2014 Feb 5;14:18. doi: 10.1093/jis/14.1.18.
The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (K(M)) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population.
2.Purification of acetylcholinesterase by 9-amino-1,2,3,4-tetrahydroacridine from human erythrocytes.
Kaya HB1, Özcan B, Şişecioğlu M, Ozdemir H. Appl Biochem Biotechnol. 2013 May;170(1):198-209. doi: 10.1007/s12010-013-0177-3. Epub 2013 Mar 15.
The acetylcholinesterase enzyme was purified from human erythrocyte membranes using a simple and effective method in a single step. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a well-known drug for the treatment of Alzheimer's disease, which inhibits cholinesterase. We have developed a tacrine ligand affinity resin that is easy to synthesize, inexpensive and selective for acetylcholinesterase. The affinity resin was synthesized by coupling tacrine as the ligand and L-tyrosine as the spacer arm to CNBr-activated Sepharose 4B. Acetylcholinesterase was purified with a yield of 23.5 %, a specific activity of 9.22 EU/mg proteins and 658-fold purification using the affinity resin in a single step. During purification, the enzyme activity was measured using acetylthiocholine iodide as a substrate and 5,5'-dithiobis-(2-nitrobenzoicacid) as the chromogenic agent. The molecular weight of the enzyme was determined as about 70 kDa monomer upon disulphide reduction by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
3.Electrochemical detection of malathion pesticide using acetylcholinesterase biosensor based on glassy carbon electrode modified with conducting polymer film.
Guler M1, Turkoglu V2, Kivrak A2. Environ Sci Pollut Res Int. 2016 Mar 16. [Epub ahead of print]
Acetylcholinesterase (AChE) biosensor based on conducting poly([2,2̍';5̍' 2″]-terthiophene-3̍-carbaldehyde) (PTT) modified glassy carbon electrode (GCE) was constructed. AChE was immobilized on PTT film surface through the covalent bond between aldehyde and amino groups. The properties of PTT modified GCE were studied using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). The biosensor showed an oxidation peak at +0.83 V related to the oxidation of thiocholine, hydrolysis product of acetylthiocholine iodide (ATCI), catalyzed by AChE. The optimum current response of the biosensor was observed at pH 7.5-8.0, 40 °C and 120 U/cm2 of AChE concentration. The biosensor showed a high sensitivity (183.19 μA/mM), a linear range from 0.015 to 1.644 mM, and a good reproducibility with 1.7 % of relative standard deviation (RSD). The biosensor showed a good stability. The interference of glycin, ascorbic acid, histidine, uric acid, dopamine, and arginine on the biosensor response was studied.
4.Critical evaluation of acetylthiocholine iodide and acetylthiocholine chloride as substrates for amperometric biosensors based on acetylcholinesterase.
Bucur MP1, Bucur B, Radu GL. Sensors (Basel). 2013 Jan 25;13(2):1603-13. doi: 10.3390/s130201603.
Numerous amperometric biosensors have been developed for the fast analysis of neurotoxic insecticides based on inhibition of cholinesterase (AChE). The analytical signal is quantified by the oxidation of the thiocholine that is produced enzymatically by the hydrolysis of the acetylthiocholine pseudosubstrate. The pseudosubstrate is a cation and it is associated with chloride or iodide as corresponding anion to form a salt. The iodide salt is cheaper, but it is electrochemically active and consequently more difficult to use in electrochemical analytical devices. We investigate the possibility of using acetylthiocholine iodide as pseudosubstrate for amperometric detection. Our investigation demonstrates that operational conditions for any amperometric biosensor that use acetylthiocholine iodide must be thoroughly optimized to avoid false analytical signals or a reduced sensitivity. The working overpotential determined for different screen-printed electrodes was: carbon-nanotubes (360 mV), platinum (560 mV), gold (370 mV, based on a catalytic effect of iodide) or cobalt phthalocyanine (110 mV, but with a significant reduced sensitivity in the presence of iodide anions).