Acetylthiocholine

S-Acetylthiocholine bromide [25025-59-6] M 242,2, m 217-223°(dec), It is a hygroscopic solid which can be recrystd from ligroin-EtOH (1 1), dried and kept in a vacuum desiccator. Crystn from C H -EtOH gave m 227° or from propan-l-ol the m was 213°. [Acta Chem Scand 11 537 1957, 12 1481 7958.]... 

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 CL resin column (prepared by washing with N HCl followed by COf— free H2O until the pH is 5.8). After equilibration for lOmin elution is started with CO3—free distilled H2O and... 

S-Acetylthiocholine iodide [1866-15-5] M 289.2, m 203-204°, 204°, 204-205°. Recrystd from propan-l-ol (or wo-PrOH, or EtOH/Et20) until almost colourless and dried in a vacuum desiccator over P2O5. Solubility in H2O is 1% w/v. A 0.075M (21.7mg/mL) solution in O.IM phosphate buffer pH 8.0 is stable for 10-15 days if kept refrigerated. Store away from light. It is available as a 1% soln in H2O. [Biochemical Pharmacology 7, 88 I96I IR Hansen Acta Chem Scand 13 151 1959, 11 537 1957 Clin Chim Acta 2 316 7957 Zh Obshch Khim 22 267 1952.]... 

Figure 3. Plot of V against total enzyme [ET] showing the irreversible inhibition of el tric eel acetylcholinesterase (AChE) by ANTX-A(S). The enzymes were incubated with 0.32 fig/mL ANTX-A(S) for 1.0 min and acetylthiocholine (final concentrations 2.5, 4.7, 6.3, and 7.8 X 10 M) was added. V was determined from the double reciprocal plots (not shown). Key (o) control ( ) ANTX-A(S). (Reproduced with permission from Ref. 42. Copyright 1987 Pergamon Press)...

Reagents Acetylthiocholine iodide or chloride (ATCh, Sigma) maleate buffer 0.1 M, pH = 6.0 sodium citrate CuS04-5H20 distilled H20 potassium ferricyanide commercial acetylcholinesterase or water extract any cholinesterase-containing animal or plant. 

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... 

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

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. 

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...

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

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

Francis placed strips of the retina from different animals in sodium sulphate to precipitate the cholinesterase in situ. Some strips were then incubated with acetylthiocholine, while others were kept in D.F.P. solution before the incubation. The tissues after preliminary washings were then treated with appropriate reagents so as to precipitate the copper derivative of thio-choline. The sections ultimately obtained showed dark deposits at those points where the enzyme was present, and deposits were absent if D.F.P. had destroyed the enzyme. As a result of the application of this technique, Francis was able to establish that for all the animals examined, except the frog, true cholinesterase was present only at the inner synaptic layer. 

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. 

The dynamic features of each of the thiols were subsequently evaluated in transthiolesterification reactions in buffered D O solution (NaOD/D PO, pD 7.0) with the ACh analog acetylthiocholine [ASCh (14), Table 6.1]. Formation/thiolysis of each thiolester was carefully followed by H-NMR spectroscopy at different time intervals, and exchange rate and equilibrium composition were determined for each combination. The rate of exchange was directly correlated to the p/f of the thiols the lower the pK, the faster the exchange reaction (Table 6.1). Thiols having pK values lower than 8.5 reached equilibrium very rapidly. The results also showed that the majority of thiols produce equilibrium concentrations that are close to... 

Selected entries from Methods in Enzymology [vol, page(s)] Acetylthiocholine as substrate, 251, 101-102 assay by ESR, 251, 102-105 inhibitors, 251, 103 modification by symmetrical disulfide radical, 251, 100 thioester substrate, 248, 16 transition state and multisubstrate analogues, 249, 305 enzyme receptor, similarity to collagen, 245, 3. 

X 10 with acetylthiocholine. On the other hand, neutral substrates such as phenylacetate and isoamyla-cetate have bimolecular rate constants of 6 x 10 and... 

Occupational air Air exposed to polystyrene strip with adsorbed cholinesterase assayed in cuvette with Ellman s reagent and acetylthiocholine UV absorbance (412 nm) 20 ppb (1-min exposure) No data Brown et al. 1984... 

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],... 

The second pK, 8.5-9.5, derived from the pH-activity curves, is much more difficult to interpret. This pK is naturally absent in the system imidazol + ester (21). It is also subject to much greater variation than pK0. This has been demonstrated for a variety of substrates (Fig. 3), but is especially prominent when thiol esters are being studied (Figs. 4 and 5). In the system eel esterase-acetylthiocholine, no decrease of activity is observed on the alkaline side up to pH 11, and for plasma cholinesterase-acetylthiocholine the decrease is very much delayed, when compared with the oxy ester, acetylcholine (see Fig. 2). Similar observations have been made with other esterases and other thiol esters (44)- They indicate that the second component 02 of the esteratic site, to which pK has to be ascribed, may be less essential for certain substrates than for others. 

Fig. 4. pH-activity curve of the system eel esterase-acetylthiocholine. After Bergmann, et al. (21). Substrate concentration 4 X 10 3 M. 

Following immobilisation, the beads were dispersed in an aqueous solution of HEC and cast onto Pt electrodes. Activity tests showed that leaching of immobilised enzyme was 2.5 times slower than that of free enzyme dispersed in HEC. Comparisons of activity to acetylthiocholine after 72 h constant operation showed a large stability enhancement for enzymes immobilised on both silica and carbon when compared to dispersion in HEC [36]. 

The enzyme immobilisation was carried out on 7,7,8,8-tetra-cyanoquinodimethane (TCNQ)-modified screen-printed electrodes. TCNQ allows the electrochemical oxidation of thiocholine, the product of the reaction between acetylthiocholine and the enzyme, at +100 mV (vs. Ag/AgCl) (Fig. 16.6). The enzymatic activity of the acetylcholinesterase can thus be monitored by electrochemical methods. 

Fig. 16.6. Schematic representation of the enzymatic reaction between acetylcholinesterase and acetylthiocholine and the subsequent thiocholine detection on the TCNQ-modified electrode surface.

OPH-based biosensors have been fully discussed in previous reviews [2,165]. AChE-based biosensors are based on the principle that OP pesticides have an inhibitory effect on the activity of AChE that may be permanent or partially reversible. The extent of the inhibition is directly related to the concentration of the pesticide and therefore enzyme activity may be used as a measure of the inhibition [166]. The amperometric measurement of AChE activity can be based on the measurement of any of the following three mechanisms [167] (1) production of hydrogen peroxide from choline, (2) oxygen consumption during the enzyme reaction or (3) production of electroactive compounds directly from the oxidation of acetylthiocholine chloride such as thiocholine. The measurement of hydrogen peroxide and oxygen consumption has been described in more details in other reviews [167]. 

Another class of enzymes that has found wide application in the biosensor field in the last decades is that of the cholinesterases which have been mainly used for the detection of pesticides. For the amperometric detection of cholinesterase activity, both the substrates acetylcholine and acetylthiocholine have been extensively used [6-9], the latter being preferred because this avoids the use of another enzyme, choline oxidase, which is usually coupled with acetylcholinesterase. However, the amperometric measurement of thiocholine, produced by... 

Scheme 1. Schematic representation of the system adopted for glucose and pesticide detection. In the upper part of the scheme is shown the reaction chain for the detection of acetylthiocholine giving a measure of acetylcholinesterase (AChE) activity which can be related to pesticide content. In the lower part of the scheme is shown the classic reaction utilised in the case of an oxidase enzyme (glucose oxidase—GOx) for the detection of glucose. In the first case, the final product is thiocholine and in the second, H202, both are measured at the Prussian blue modified electrode at an applied potential of 0.2 V vs. Ag/AgCl and —0.05 V vs. Ag/AgCl, respectively.

The inhibition and the subsequent signal detection were performed in two different solutions. First the pesticide solution was added and then after 10 min (incubation time) the sensor was moved into a new buffer solution where the substrate (5mmoll 1 acetylthiocholine) was injected and the signal measured. This procedure is particularly suitable when a complex matrix, which could pose problems for the direct measurement of thiocholine oxidation, is used. The analytical characteristics of pesticide determination in standard solutions were then evaluated. Detection limits, defined in this work as the concentrations giving an inhibition of 20%, were 30 and 10 ppb for aldicarb and paraoxon, respectively. By increasing the incubation time up to 30 min, an increase in the degree of inhibition could be observed and lower detection limits both for Aldicarb (5 ppb) and Paraoxon (3 ppb) were achieved. 

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