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Introduction
receptors. This action must be regulated to avoid over-stimulation of, for instance, the
nervous system (Brown, 2006). Hence, metabolism of these chemicals through hydrolysis by
understanding of the substrate selectivity of these cholinesterases and how their hydrolysing
examined first. These enzymes differ in selectivity for substrates based on structure and
suxamethonium (Dale et al., 2007) were added to these enzymes. The hydrolysis of these
cholinergic substrates produces acetic acid. The subsequent increase in pH was modelled
demonstrated metabolism, with elevated rates of colour change denoting increased selectivity
of the enzyme for the substrate. The action of acetylcholinesterase on acetylcholine and
of inactive complexes of varying stabilities (Burgen, 1949). Hence, the effects of the
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inhibitors edrophonium (short-acting), physostigmine and neostigmine (medium-acting), and
observed (Dale et al., 2007). Atropine and carbachol action, a cholinergic antagonist and
The aims of this practical were to demonstrate the selectivity of acetylcholinesterase and
velocity (RV) of colour change would be observed when substrates are in the presence of a
cholinesterase for which they are more structurally selective. Furthermore, the RV was
anticipated to decrease as the duration of action of the inhibitor specific for the cholinesterase
in use increased.
Results
195.0
200.0
180.0
160.0
140.0
120.0
100.0 100.0
100.0
80.0
60.0
40.0
Relative Velocity (%)
20.0
0.0 0.0 2.6 1.1 0.0 0.0
0.0
1 2 3 4 5 6 7 8 9
Condition
presence of water and Tris buffer, in condition (1) 100 mM acetylcholine was added
to water and mixed, while in the remaining conditions various 100 mM substrates or
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water were individually added to acetylcholinesterase – condition (2) water; (3)
(8) methacholine; (9) suxamethonium. Both the degree and time of colour change
were recorded, which were used to calculate the relative percentage velocity of
colour change of the each condition relative to the mean velocity of colour change of
conditions 3 and 4.
condition 1 (acetylcholine with water) and condition 2 (water with acetylcholinesterase) did
120.0
104.2
100.0 100.0
100.0
80.0
60.0
40.0 33.8
21.9
20.0
Relative Velocity (%)
0.0 0.0 1.3 0.0
0.0
1 2 3 4 5 6 7 8 9
Condition
presence of water and Tris buffer, in condition (1) 100 mM Butyrylcholine was added to
water and mixed, while in the remaining conditions various 100 mM substrates or water
were individually added to ButyrylcholineE – condition (2) water; (3) butyrylcholine; (4)
butyrylcholine; (5) acetylcholine; (6) benzoylcholine; (7) carbachol; (8) methacholine; (9)
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suxamethonium. Both the degree and time of colour change were recorded, which were
used to calculate the relative percentage velocity of colour change of the each condition
condition 2 (water with butyrylcholinesterase) did not produce any colour change.
160.0
139.3
140.0
121.9
120.0
100.0
80.0
60.0
0.0
Physostigmine Neostigmine Edrophonium Malathion Carbachol
(10mM)
Inhibitor
added to Tris buffer and Acetylcholinesterase, and 100 mM acetylcholine was then added
to this mixture. Both the degree and time of colour change were recorded, which were
used to calculate the relative percentage velocity of colour change of the each condition
acetylcholinesterase.
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The RV for the hydrolysis of acetylcholine by acetylcholinesterase (100%) was increased to
139.3% in the presence of 50mM malathion, and to 121.9% in 5mM atropine (Figure 3). It
was, however, decreased in 80mM edrophonium (25.0%), 10mM carbachol (17.1%), 5mM
120.0 113.6
100.0
89.3 89.3
80.0
60.0
40.0
were added to Tris buffer and butyrylcholinesterase, and 100 mM butyrylcholine was
then added to this mixture. Both the degree and time of colour change were recorded,
which were used to calculate the relative percentage velocity of colour change of each
condition relative to the mean velocity of colour change of butyrylcholine in the presence
of butyrylcholinesterase.
increased in the presence of 50mM malathion (113.6%). It was, however, decreased in the
presence of 80mM edrophonium (89.3%), 5mM atropine (89.3%), 10mM carbachol (9.8%),
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Discussion
and butyrylcholinesterase and also to study the inhibitory effects of various short, medium
and long acting cholinesterase inhibitors. The results supported our first hypothesis that
certain substrates would be more selective for either cholinesterase and that this would be
visualized through higher relative velocity of colour change. Our second hypothesis that
longer acting inhibitors would result in subsequently lower RV was also partially supported.
In the current study, the RV of acetylcholine was considerably higher in the presence
substrates.
This enzyme specificity for certain substrates is due to the active site of
butyrylcholinesterase favouring longer alpha carbon chains and functional groups and the
For the same reasons, it was expected that benzoylcholine would be hydrolysed more
the alpha carbon of this substrate. The current study confirmed this, with an RV of 104% in
noted that benzoylcholine should still be hydrolysed slower than the control substrate
butyrylcholine due to a lower affinity to the binding site as described by Fraser (1956). It is
likely that inaccurate subjective human perception of colour change affected the result.
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Similarly, suxamethonium is known to be hydrolysed by butyrylcholinesterase but not
Acetylcholinesterase due to the deep gorge in the active site within Butyrylcholinesterase
(Saxena et al., 1997). However, experimentally, it was observed that suxamethonium was not
the Butyrylcholinesterase gene that translated to mutations in the binding site as described by
acetylcholinesterase (195%, Figure 1), which was unexpected. Indeed methacholine should
not be rapidly hydrolysed by acetylcholinesterase due to the steric hindrance caused by the
proximity of the methyl group to the ester group resulting in a lower rate of hydrolysis
(Bruning et al., 1996; Foye et al., 2008). For the same reason, methacholine should also resist
(2008) obtained similar results, and found that the hydrolysis of the carbamolyated enzyme
intermediate after the initial enzyme-carbachol complex is rate limiting. The slow hydrolysis
was expected and confirms that carbachol is not specific for either cholinesterase.
Overall the results showed that neither enzyme was more active than the other,
however, it was demonstrated that substrates with larger and smaller functional groups were
The second part of the experiment, revealed the inhibitory actions of various
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(Cook, 1992). The medium acting cholinesterase inhibitors physostigmine and neostigmine,
inhibited Acetylcholinesterase, with RVs of 7.4% and 10.5% respectively (Figure 3).
It has been previously demonstrated that stigmines exert a greater inhibition compared
to shorter acting inhibitors such as edrophonium (Sakuma et al., 1992) Edrophonium, binds
electrostatically to the anionic site of Acetylcholinesterase and to the esteric site by hydrogen
bonding. The stigmines also bind electrostatically to cholinesterases, but produce longer
inhibition due to the formation of a strongly bound covalent carbamylated intermediate at the
esteric site (Riviere and Papich, 2009). This intermediate resists hydrolysis. Thus, even
though initial inhibitor concentration of the stigmines (5uM) was 16 times less than
edrophonium (80uM) the effect was greater. This confirmed part of our second hypothesis.
Furthermore, it can be seen that even at low concentrations (5-80uM), inhibitors can still
outcompete substrates at greater concentrations (100mM) for the cholinesterase binding site.
al., 2007) would be expected to produce greater inhibition than edrophonium and the
disagreeing with the second hypothesis. An explanation could be that the malathion
concentration (50uM) was insufficiently high to inhibit cholinesterase. Indeed, a study by Pei
was 10 and 1000 fold greater than edrophonium and neostigmine respectively.
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(1971) demonstrated this effect, showing that the deacetylation step in cholinesterase
hydrolysis would be aided by atropine. However, atropine is not known to interact with
laboratory conditions. These same changes likely contributed in part to the unexpected results
Butyrylcholinesterase (9.77%, Figure 4). A more likely explanation is that the high
The experiment had several problems. Firstly it was assumed that human detection of
visual change is accurate. Given some unexpected results, the use of a spectrophotometer is
each substrate and inhibitor was used. Future direction points towards utilising a range of
both the reliability and clinical applicability of the results. Laboratory conditions can also be
Furthermore, the action of both short and medium acting inhibitors was clearly elucidated.
However, the hypotheses were only partially supported, with malathion returning unexpected
responses. Clear flaws in methodology may rectify these problems, and future direction
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