Introduction: Enzymes are highly specific biological catalysts (proteins) that influence the rate of metabolic reactions (Smith, 1997). They are responsible for efficiently converting substrate molecules into products required to sustain life (Grisham et al. , 1999). The activity of enzymes can often be effected by other endogenous or foreign factors (e. g. inhibitors). Inhibitors are able to decrease the rate of enzymatic activity by binding to the enzyme and preventing it from functioning optimally.
(Hostettmann et al. , 2006). Inhibitors occur both naturally (i. e.plant and animal poisons) and as artificially synthesized substances (i. e. insecticides such as malathion) (Walsh et al. , 2011). They are also common in pharmocology and biochemistry, as many drugs are essentially enzyme inhibiting molecules used clinically to help treat conditions such as glaucoma, myasthenia gravis and Alzheimer’s disease (Scapin, 2006; Liston et al. , 2004).
In this particular experiment, we are attempting to calculate the rate of the reaction (hydrolysis) of different substrates by enzymes acetylcholinesterase and butyrylcholinesterase, in the presence of various inhibitors (anticholinesterases).
Acetylcholinesterase (or erythrocyte cholinesterase) is mainly found in red blood cells, neural synapse, cholinergic nerve fibres and muscle motor end-plates (Wang & Tang, 2005). Butyrylcholinesterase (or pseudocholinesterase) is localized in the plasma and liver, and unlike AChE prefers to hydrolise butyrylcholine, benzoylchline, procaine and suxamethonium more so than acetylcholine (Brash, 2009). For the purposes of this experiment, activity of the enzymes will be measured based on visual observation of the change in colour of phenol red in a dilute buffer containing sodium diethylbarbiturate.
The characteristic liberation of acid can be linked to this change in colour (decrease in basic from of the buffer) and hence it is possible to qualitatively track the hydrolysis of the substrate into its products. For example, the hydrolysis of acetylcholine to choline and acetic acid as seen below: [pic] The substrates used in this experiment are: acetylcholine, methacholine, carbachol, benzoylcholine and suxamenthonium. The inhibitors being tested are: physostigmine, neostigmine, malathion, endrophonium, atropine and carbachol. Method:
Refer to method in pharmacology 1 laboratory manual (pg. 13-14) Amendments: – (pg. 14/PartA/Step 1) 14 test tubes are required (not 13) – (pg. 14/PartA/Step 5) Transfer 250 ul of each sample to the ELISA plate (not 200 ul). Results: AChE/BChE Activity (w/Substrates) | |AChE velocity (%) (time to |Class Average |BChE velocity (%) (time to|Class Average | | |100% response) | |100% response) | | |Acetylcholine |70 |60 |435 |412. 5 | |Carbachol |1. 7 |13. 8 |40 |54 | |Methacholine |215 |192. 5 |71 |76 | |Benzoylcholine |17 |28. 85 |721 |510. 5 | |Suxamethonium |0 |7. 4 |27* |47.
5* | Table 1: Time taken for AChE/BChE to achieve 100% response (colour change) in the presence of various substrates (w/o inhibitors). *= Unexpected Result Table 1 displays the data acquired from the reaction of AChE/BChE with five different substrate molecules. Methacholine had the highest AChE velocity and benzoylcholine had the the highest for BChE, indicating that they reached the colour change quicker than any of the other substrates in the presence of their respective enzymes. Surprisingly, suxamethonium yielded the lowest values of relative velocity for both enzymes.
This result is unexpected and tends to skew the accuracy of this set of results. This is further evident in Figure 1, below. [pic] Figure 1: Time taken for AChE/BChE to achieve 100% response (colour change) in the presence of various substrates (graphical representation of Table 1). AChE/BChE Activity (w/Inhibitors) | |AChE velocity (%) (time to |Class Average |BChE velocity (%) (time to|Class Average | | |100% response) | |100% response) | | |Physostigmine |1579 |1339. 5 |4 |51 | |Neostigmine |910 |845 |100 |87. 5 | |Endrophonium |258 |209 |785 |722.
5 | |Malathonion |110 |90 |630 |765 | |Malaoxon |1610 |1460 |620 |790 | |Atropine |123 |94. 5 |500 |700 | |Carbachol |510 |445 |1170 |1515 | Table 2: Time taken for AChE/BChE to achieve 100% response (colour change) in the presence of various inhibitors. Table 2 represents the data obtained from the reaction of AChE/BChE in the presence of seven different inhibitors. Malathonion and physostigmine showed the lowest values for relative velocity for AChE and BChE respectively. This indicates their effectiveness in inhibiting the enzymes and increasing the time required to reach colour change.
Inversely, the samples containing malaoxon (AChE) and carbachol (BChE) were the two highest values for velocity, meaning they were the samples to reach the required colour change the quickest. [pic] Figure 2: Time taken for AChE/BChE to achieve 100% response (colour change) in the presence of various inhibitors (graphical representation of Table 2). In this experiment, relative velocity was determined by calculating the velocity of each of the samples as the percentage of the velocity of the control sample (completely yellow sample).
The equations (from pharmacology 1 laboratory manual pg. 14) below were used: [pic] % change = (Absorbance of test reaction x 100)/ Absorbance of control Note: All times are in seconds. For example: Carbachol/AChE relative velocity % = (4257 x100)/ 250411 = 1. 7 (b) physostigmine/AChE relative velocity % = (4257x 0. 241)/ 0. 649 = 1579 Discussion: For the most part, there was little significant deviations in the results from previously established literature on this topic.
The one most substantial and unexpected of these deviations was that of the substrate suxamethonium. It is widely known and accepted that butyrylcholinesterase is favorable towards the hydrolysis of suxamethonium (Gennaro et al. , 2006; DiPiro & Joseph, 2005). Yet the results achieved from this experiment contradict such knowledge, as seen in Table 1- average of 47. 5 BChE velocity. This may be due to a human error/contamination during the experimental procedure itself or a problem with the suxamethonium supplied for this experiment.
The latter seems to be more likely, as all the groups that attempted this experiment yielded similar low numbers for suxamethonium. Discounting the unusual results for suxamethonium, it was found that benzoylcholine and carbachol were the best and worst substrates respectively; and atropine and malaoxon were the best and worst inhibitors respectively Due to this unexpected result, it is advisable that the experiment be repeated multiple times with different batches of suxamethonium in order to confirm that this was merely caused by a problem with the sample.
Furthermore, the majority of studies done regarding this topic are quantitate measures of cholinesterase activity, making it difficult to effectively compare the results of such experiments with this one (Amitai et al. , 1998; Liston et al. , 2004). However this does not cheapen the value of such experiments , but indeed points to the need for further studies such as this to be done in order to increase the reliability and validity of the results obtained. [pic] Acetylcholine Acetic Acid Choline The reaction above shows a common example of a cholinesterase hydrolysis reaction- hydrolysis of acetylcholine by acetylcholinesterase.
The active site of AChE comprises 2 subsites – the anionic site and the esteratic subsite. The structure and mechanism of action of AChe are closely related. The anionic subsite accommodates the positive quaternary amine of acetylcholine as well as other cationic substrates and inhibitors. The cationic substrates are not bound by a negatively-charged amino acid in the anionic site, but by interaction of 14 aromatic residues that line the gorge leading to the active site.  Up to question 2 in lab manual questions. Conclusion:
In this experiment we aimed to calculate the rate of the reaction of different substrates by acetylcholinesterase and butyrylcholinesterase in the presence of different inhibitory substances. This was done by visually observing the degree of activity from the change in colour of phenol red in dilute buffer of sodium diethyl-barbiturate. It was found that benzoylcholine and carbachol were the best and worst substrates respectively; and atropine and malaoxon were the best and worst inhibitors respectively. (This is excluding the results for suxamethonium). Bibliography:
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