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Drugs

Cholinesterase and its inhibitors

acetylcholine chloride atropine sulphate benzoylcholine chloride carbachol chloride edrophonium chloride malathion (dissolved in 0.1% DMSO) malaoxon methacholine chloride neostigmine bromide physostigmine salicylate suxamethonium chloride butyrylcholinesterase (horse) true acetylcholinesterase (eel erythrocytes) sodium diethylbarbiturate solution buffer indicator 10 Units/ml 100 mmol/l 5 mol/l 100 mmol/l 100 mmol/l 50 mol/l 50 mol/l 50 mol/l 100 mmol/l 50 mol/l 50 mol/l 100 mmol/l

Introduction
In the mammal there are at least two types of enzyme which hydrolyse acetylcholine to choline and acetic acid: (CH3) 3N+.CH2.CH2.O.CO.CH3 + H2O ! (CH3) 3N+.CH2.CH2.OH + CH3.CO.OH True cholinesterase (acetylcholinesterase: AChE) occurs in red blood cells, in cholinergic nerve fibres and in muscle (motor end-plates). Butyrylcholinesterase (pseudocholinesterase or serum cholinesterase; BChE) occurs in plasma and is probably produced in the liver. The two enzymes differ in a number of respects, namely their distribution, substrate specificity, and functions. Butyrylcholinesterase hydrolyses butyrylcholine more rapidly than acetylcholine, as well as other esters such as benzoylcholine, procaine and suxamethonium. In this experiment, the substrates used are acetylcholine, acetyl-"-methylcholine (methacholine) carbamylcholine (carbachol), benzoylcholine and suxamethonium. The substances tested for inhibiting action are physostigmine (eserine), neostigmine, malathion, edrophonium, atropine and carbachol. Inhibitors (anticholinesterases) are used clinically to increase the tone and motility of the intestine and bladder in atonic states, to constrict the pupil and reduce intra-ocular pressure in glaucoma, and to improve skeletal muscle function in myasthenia gravis. Anticholinesterases can be considered as short-acting, medium-duration, or irreversible.

Method
Nearly all of the methods for measuring cholinesterases are based on the fact that acid is liberated by the hydrolysis of acetylcholine. In the present experiment, the degree of activity is estimated visually from the change in colour of phenol red in a dilute buffer of sodium diethylbarbiturate.

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Since the indicator and diethylbarbituric acid have about the same pKa (7.6-7.7), a decrease in the red (alkaline) form of the indicator accurately reflects any decrease in the basic form of the buffer, which in turn decreases stoichiometrically with the enzymatic liberation of acid. (Quantitative measurements of the enzyme can be accomplished with a colorimeter or spectrophotometer to measure the colour change.) PROCEDURE Part A: True acetylcholinesterase A preparation of true acetylcholinesterase derived from eel red blood cells will be available. The reactions are performed at room temperature. NOTE: Do not insert pipettes into class stock solutions of buffer, enzymes, inhibitors or substrates, but pour from the stock bottles into clean dry containers, slightly more than you will need: 40 ml buffer indicator solution, 7.5 ml acetylcholine, and less than 1 ml of each of the other solutions. Rinse pipettes thoroughly with water and shake as dry as possible before using in a different solution; slight contamination, particularly with one of the powerful inhibitors, might spoil the experiment. Any necessary dilutions of inhibitor or substrate stock solutions should be made with distilled water. 1. Arrange a series of 13 test tubes, each containing 1.1 ml of sodium diethylbarbiturate buffer indicator solution (2 mmol/l sodium diethylbarbiturate, 0.15 mmol/l HCl, 8 !g/ml phenol red), and add the amounts of enzyme and inhibitor (or water) indicated in Table 1. Appropriate personal protection must be worn when pipetting (goggles and gloves). Mix well by tapping. Do not mix by shaking, since this could favour the entry of carbon dioxide from the air into the samples. 2. The reactions are initiated by the addition of substrates (or water) as wet out in Table 1, and mixing by tapping (do not shake). In the samples containing inhibitors (numbers 8 to 13), incubate for 20 min at room temperature before adding substrate. In the case of malathion (number 11) incubate for 10 min before adding substrate. This allows adequate time for reaction between the enzyme and the inhibitor. 3. Perform the assays on samples 1 to 3 first, so that the expected colour changes can be gauged, and the standard time to complete yellow can be measured. It is then suggested that the subsequent assays be initiated by addition of substrate at 1 min intervals. Make certain that adequate incubation of enzyme and inhibitor has been provided.

4. Enter your results in Table 1 and, when they are calculated, transfer the relative velocities to the "Summary of Results of Acetylcholinesterase Inhibition" sheet. Record the time in min at which each sample becomes nearly completely yellow. Try to use the same end-point for all (use tube 3 as a standard against which to check the completeness of reaction). 5. In the case of samples which are still pink or red 60 min after initiating the reaction, estimate the approximate percentage change from red to yellow (0, 25, 50, 75%). This can be achieved by transferring 200 !l of each sample to a 96-well ELISA plate and reading the absorbance at 460 nm. For this purpose use the blank without enzyme as 0% change (sample 1) and completely yellow sample (sample 3) as 100% change. Calculate the velocity of the samples as the percentage of the velocity of control sample 3 as follows: for those which go to completion within 60 min:

relative velocity % =

time for sample 3 ! 100 time for observed sample

for those which are not complete at 60 min:

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relative velocity % =

time for sample 3 ! %change 60

Part B: Butyrylcholinesterase (pseudocholinesterase or serum cholinesterase) A preparation of butyrylcholinesterase from horse serum will be available. There may be differences in specificity between this horse serum cholinesterase and human serum cholinesterase. A total of 13 samples are tested as indicated in Table 2, in the same manner as that described in Part A. Enter these results in Table 2 and, when they are calculated, transfer the relative velocities to the "Summary of Results of Acetylcholinesterase Inhibition" sheet.

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Questions
1. Provide the complete chemical reactions for hydrolysis of ACh by AChE. Show all intermediary steps. 2. Give the chemical structures for the substrates used in this experiment. Using these structures try and explain the substrate specificity of the cholinesterases 3. How does the initial concentration of substrate compare with the concentration of the inhibitor in the various samples? 4. Compare and contrast the results obtained with true acetylcholinesterase and butyrylcholinesterase. Can any differences be explained in terms of difference in molecular structure of these two enzymes. 5. From the results, can it be definitely said that one enzyme is more active than the other, or is additional information required?

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TABLE 1: True acetylcholinesterase

If Time to incomplete Sample AChE complete at 60 min Inhibitor or Substrate or yellow or No. estimated % water (0.3 ml) water (0.3 ml) (min) water change (0.8 ml) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 water AChE AChE AChE AChE AChE AChE AChE AChE AChE AChE AChE AChE AChE water water water water water water water physostigmine 50 mol/l neostigmine 50 mol/l edrophonium 50 mol/l malathion 50 mol/l malaoxon 50 mol/l atropine 5 mol/l *carbachol 10 mmol/l acetylcholine 100 mmol/l water acetylcholine 100 mmol/l carbachol 100 mmol/l methacholine 100 mmol/l benzoylcholine 100 mmol/l suxamethoniu m acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l X X X 100 0 Additions to the 1.1 ml buffer indicator Velocity relative to sample 3 (%) 0

* Dilute stock solution with water. AChE = true acetylcholinesterase.

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Table 2: Butyrylcholinesterase
If Time to incomplete Sample BChE complete at 60 min Inhibitor or Substrate or yellow or No. estimated % water (0.3 ml) water (0.3 ml) (min) water change (0.8 ml) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 water BChE BChE BChE BChE BChE BChE BChE BChE BChE BChE BChE BChE BChE water water water water water water water physostigmine 50 mol/l neostigmine 50 mol/l edrophonium 50 mol/l malathion 50 mol/l malaoxon 50 mol/l atropine 5 mol/l *carbachol 10 mmol/l acetylcholine 100 mmol/l water acetylcholine 100 mmol/l carbachol 100 mmol/l methacholine 100 mmol/l benzoylcholine 100 mmol/l suxamethoniu m acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l acetylcholine 100 mmol/l X X X 100 0 Additions to the 1.1 ml buffer indicator

Velocity relative to sample 3 (%) 0

* Dilute stock solution with water. BChE = Butyrylcholinesterase.

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Summary of Results for Acetylcholinesterase Inhibition Substrates

AChE velocity Class Av. (%) (time to 100% response) acetylcholine carbachol methacholine benzoylcholine suxamethonium BChE velocity Class Av. (%) (time to 100% resp)

Inhibitors
AChE velocity Class Av. (%) (time to 100% response) physostigmine neostigmine edrophonium malathion atropine carbachol BChE velocity Class Av. (%) (time to 100% resp)