Acetylthiocholine hydrolysis

Principle The determination of acetylthiocholine hydrolysis, based on the absorbance at 412 nm of the yellow product - complex of thiocholine with Ellman reagent. 

Fig. 4 Formulae of alkaloids wh Table 1. The rate (V) of the acetylthiocholine hydrolysis by water extracts (1 10 weight/volume) Equisetum arvense in the presense of allelochemicals -alkaloids and ozone...

As proof of principle, Lehn and coworkers individually synthesized all acyl hydrazone combinations from the 13 DCL building blocks and measured their inhibition of acetylthiocholine hydrolysis by ACE in a standard assay. They then established a dynamic deconvolution approach whereby the pre-equilibrated DCL containing all members is prepared, frozen, and assayed. Thirteen sublibraries were then prepared containing all components minus one hydrazide or aldehyde component, and assayed. Active components in the DCL were quickly identified by an increase in ACE activity, observed in sublibraries missing either hydrazide 7 or dialdehyde i, pointing to the bis-acyl hydrazone 7-i-7 as the most likely active constituent. This was in line with the individual assay data recorded earlier resynthesis of this compound characterized it as a low nanomolar inhibitor of the enzyme. 

Observations The hydrolysis of acetylthiocholine during 1 h leads to the formation of red-brown coloured product, which can be seen without any technique (see Experiment 1) or measured by photometric technique in... 

Observations The reaction lasts 1 h. The results are expressed in pM s 1kg" 1 of fresh mass. As shown in our experiments, the rates of hydrolysis of acetylthiocholine of studied plant cells varied from 0.4 0.05 pM s 1kg 1 of fresh mass for pollen of knights star up to 0.105 0.01 pM s 1kg 1 of fresh mass for horsetail vegetative microspores. Table 1 shows I50 for inhibition of cholinesterase from microspores by used inhibitors. 

Photometric measurements of colouring in the AChE-biotests after biochemical hydrolysis of acetylthiocholine may be made by using any... 

S-Acetylthiocholine chloride [6050-81-3] M 197.7, m 172-173" The chloride can be purified in the same way as the bromide, and it can be prepared from the iodide. A few milligrams dissolved in H2O can be purified by applying onto a Dowex-1 Cf resin column (prepared by washing with N HCl followed by CO3— free H2O until the pH is 5.8). After equilibration for lOmin elution is started with CO3 —free distilled H2O and 3ml fractions are collected and their OD at 229nm measured. The fractions with appreciable absorption are pooled and lyophilised at 0-5°. Note that at higher temps decomposition of the ester is appreciable hydrolysis is appreciable at pH >10.5/20°. The residue is dried in vacuo over P2O5, checked for traces of iodine (cone H2SO4 and heat, violet vapours are released), and recrystd from propan-l-ol. [Clinica Chim Acta 2 316 1957],... 

Chemiluminescent probes based on the dioxetane moiety are now being developed for the detection of cholinesterase activity <2002JA4874>. In this particular case, the nucleophilic thiol generated from hydrolysis of acetylthiocholine iodide triggers the formation of the thiolate 61 and by-product 62 through nucleophilic attack on the disulfide bond of 60 (Scheme 14). 

Patocka, J., Bajgar, J., Bielavsky, J. (1973). Kinetics of hydrolysis of acetylthiocholine by oximes. Coll. Czechoslov. Chem. Commun. 38 3685-93. 

In this section, we discuss about the screen printed electrode (SPE) based AChE sensors for the selective determination of OP and CA pesticides. In the past decades, several attempts were made by the researchers to develop SPE based pesticide sensors, where the enzyme AChE was immobilized either directly onto the electrode or above other matrices incorporated SPE surfaces. Both approaches resulted in the good, rapid detection of OP and CA pesticides. Earlier, Hart et al. employed AChE/SPE to detect OP and CA pesticides [21], They measured the enzyme activity from the rate of hydrolysis of acetylthiocholine iodide. Three polymers such as hydroxyethyl cellulose, dimethylaminoethyl methacrylate, and polyethyleneimine were used as enzyme immobilization matrices. Initially, electrodes were exposed to drops of water or pesticide solution, dried and their activity was screened after 24 h. They found that, when the enzyme matrix was hydroxyethyl cellulose, electrode activity inhibited both by water as well as by pesticides. While with co-polymer matrix, a significant response towards pesticides alone was observed. Further, the long-term storage stability of electrodes was highest when the enzyme matrix consisted of the co-polymer. The electrodes retained their activity for nearly one year. In contrast, the electrodes made of hydroxyethyl cellulose or polyethyleneimine possess less stability. 

As mentioned in the previous section, the response, the stability and the enzyme activity found greatly enhanced at the MWCNT platform. Other than CNTs, AuNPs also possess some unique properties and recent years it has been widely employed in the biosensors to immobilize biomolecules. Thus in this section we discuss about the application of AuNP matrix for the immobilization of AChE for pesticide sensor development. With the use of AuNPs, the efficiency and the stability of the pesticide sensor gets greatly amplified. Moreover, the nanoparticles matrix offers much friendly environment for the immobilized enzyme and thus the catalytic activity of the enzyme got greatly amplified. Interestingly, Shulga et al. applied AChE immobilized colloidal AuNPs sensor for the nM determination of carbofuran, a CA pesticide [16], The enzyme-modified electrode sensor was also utilized for the sensitive electrochemical detection of thiocholine from the enzyme catalyzed hydrolysis of acetylthiocholine chloride (ATCl). The fabrication and the enzyme catalyzed reaction at the AuNPs coated electrode surface is shown in Fig. 6. 

Acetylcholinesterase (AChE) (also termed true cholinesterase ) is found in the synaptic cleft of cholinergic synapses, and is of undoubted importance in regulation of neurotransmission by rapid hydrolysis of released endogenous acetylcholine (ACh). AChE is also found in erythrocytes and in the CSF, and can be present in soluble form in cholinergic nerve terminals, but its function at these sites is not clear, AChE is specific for substrates that include acetylcholine and the agents methacholine and acetylthiocholine. but it has little activity with other esters. It has a maximum turnover rate at very low concentrations of AChE (and is inhibited by high concentrations). 

Theoretical Modeling of Enzymic Hydrolysis of Acetylcholine Compared to Acetylthiocholine... 

The colorimetric method is based on the hydrolysis of the substrate acetylthiocholine to acetate and thiocholine as performed by the cholinesterase. Thiocholine is then reacted with 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) to form a yellow anion (5-thio-2-nitrobenzoate). The latter is quantitated by spectrometric analysis at 405 nm, with the concentration being proportional to the cholinesterase activity in the given sample. Also for a few days postmortem the cholinesterase activity in different tissues is measurable. ... 

The cholinesterases, as indicated by the name, hydrolyze the esters of choline. However, the specificity for choline esters is not absolute, as evidenced by the hydrolysis of other esters, albeit at a slower rate. While acetylcholinesterase has high specificity not only for acetylcholine— CH3C00(CH2)2N+(CH3)3—but also for acetylthiocholine and other acetyl esters, most plasma cholinesterases catalyze the hydrolysis of butyrylcholine, butyrylthiocholine, and other butyryl esters at faster rates than the corresponding acetyl derivatives. With some homologous esters of acetylcholine, it was shown early on (DIO) that butyrylcholine... 

A number of papers describe techniques for the determination of choline esterase activity based on amperometric measurement of the product formed as a result of enzymatic hydrolysis. The oxidation potential of choline is too high to be electro-chemically oxidized directly. In this case, artificial (nonnative) choline esterase substrates are used. Thus, butyiyl or acetyl thiocholine forms thiocholine as a result of choline esterase hydrolysis. When the analyte is not present in the solution, the substrate acetylthiocholine is converted into thiocholine and acetic acid. Thiocholine is oxidized by the appUed voltage. In the presence of an inhibitor, conversion of acetylthiocholine is decreased or even null. Furthermore, the anodic oxidation current is inversely proportional to the concentration of pesticides in samples and the exposed time as well. The procedure of the preparation of an AChE biosensor and pesticide detection is shown in Fig. 13.4. 

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