The aim of the present study was to determine the substrate specificity of AChE and BChE by testing these cholinesterases in the presence of a number of cholinergic compounds. Also to investigate the sensitivity of AChE and BChE to various cholinesterase inhibitors. Referring to the results of this experiment, it demonstrated that AChE had limited substrate specificity, whereas BuChE had extensive substrate specificity. BuChE hydrolysed BuCh faster when compared to Ach. It also hydrolysed other esters such as suxamethonium and neostigmine. It is evident that hypothesis for this research are accepted, and the substrate specificity of both compounds can be determined in cholinergic compound and the sensitivity in various cholinesterase inhibitors.
George and Balasubramanian’s (1981) the aryl acylamidases and their relationship to cholinesterases in human serum, erythrocytes and liver study concluded that liver and aryl acylamidase when compared with erythrocyte aryl acylamidase was associated with acetylcholinesterase. Erythrocyte and serum aryl acylamidase illustrated some similarities in their sensitivities to amine compounds like serotonin and choline derivatives, the liver enzyme had no effect to any of these compounds. The liver aryl acylamidase differed from cholinesterase inhibitor. This is evident in other researchers work e.g. Miao et al., 2010. Human serum when compared to horse serum cholinesterase showed the differences in specificity. It showed that horse serum hydrolysis with BuCh and is more potential inhibitor (Kamarić, 1975). Similar study which was carried by Eddleston et al., (2008) who hypothesised the usefulness of admission BuChE activity, together with plasma OP concentration for predicting death with two-particular OP insectides (Kolbezen et al., 1954) concludes that plasma BuChe activity can be used to predict death when the insectide ingested is known and its sensitivity and specificity for that insectide has been researched (Eddelston et al., 2008). Kolbenzen et al., (1954) study further suggests insecticidal activity of carbamate cholinesterase inhibitor and certain compounds such as protigmine, physostigmine are very effective inhibitors of insect cholinesterases. The study was based on lipid-soluble carbamates which were synthesised and evaluates contact insecticides and as in vitro-inhibitors of fly brain cholinesterase. Toxic compounds such as N-methylcarbamates, carvacol and many more were tested on green citrus aphid and the citrus red mite. They concluded that cholinergic activity and toxicity are inversely dependent in the rates of hydrolysis of carbamates and vitro-cholinesterase inhibition was related to toxicity. The second-order of hydrolysis showed cholinesterase inhibition for certain compounds (Kolbenzen et al., 1954). Jann et al., (2002) study based on clinical pharmacokinetics and pharmacodynamics of cholinesterase inhibitor suggest that AChE only binds to specific substrates where as BuChE binds to many. BuChe hydrolyses to BuCh and other ester compounds. Therefore, hypothesis and results for the current study are valid when compared with past studies. It is evident that BuChe has broad substrate specificity when compared to AChE in cholinergic compounds. The sensitivity of AChE and BChE to various inhibitors shows that BChE is more potential inhibitor than AChE. This proves our research and findings are correct.
The strength of the study was the way the experiment was carried out based on past studies and findings which correspond to present study. This proves that this research was valid and all errors that were made in past were not in present. The method of this research was completely different as to avoid errors such as human errors and misinterpretations. However, the weakness of this study was in experimental method. The tubes were not labelled and the racks were labelled. This made the procedure and keeping up with the record hard as it was difficult to see the labels/numbers on the rack and they were very small in writing. We had to also ensure the rack plate was oriented in certain position to avoid mistakes but we ended up making mistakes and confusing ourselves. This made the experimental procedure bit difficult for us and it was hard to distinguish which was substrate buffer with indicator pre-mix and enzyme-inhibitor premix. Another limitation was that we only used colometric assay spectrophotometer to measure cholinesterase activity. We could have used combination different techniques such as chromatography, co-migration of gel electrophoresis, parallel inhibition by typical cholinesterase which was used in George and Balasubramanian (1981) study. This could have given us different form of results and data which we could compare and see if we get the same results by using different techniques or not. In Kolbezen et al., (1954) study carried out more than one experiment to conclude that AChE and BuChe inhibit certain compounds in inhibitors and their cholinergic activity is dependent on hydrolysis of compound in contact to toxicity, when compared to present study it was only carried out one experiment and came to a conclusion so in future to make study much more reliable, it should have multiple tests with different techniques to measure cholinesterase activity. This would give researchers more detailed information. In conclusion, the substrate specificity of BChE is extensive compared to AChE which has limited substrate specificity in cholinergic compounds. The sensitivity of BuChE shows that BuChE hydrolyses with BuCh quicker and hydrolyses with other esters when compared to Ach.
References
1. Eddleston, M., Eyer, P., Worek, F., Sheriff, M. R., & Buckley, N. A (2008). Predicting outcome using butyrylcholinesterase activity in organophosphorus pesticide self-poisoning. Qjm, 101(6), 467-474.
2. George, S. T., & BALASUBRAMANIAN, A. S (1981). The aryl acylamidases and their relationship to cholinesterases in human serum, erythrocyte and liver. European Journal of Biochemistry, 121(1), 177-186.
3. Jann, M. W., Shirley, K. L., & Small, G. W (2002). Clinical pharmacokinetics and pharmacodynamics of cholinesterase inhibitors. Clinical pharmacokinetics,41(10), 719-739.
4. Kamarić, L. M. (1975). The inhibition by d-tubocurarine of horse serum cholinesterase-catalyzed hydrolysis of butyrylcholine and benzoylcholine.Biochemical pharmacology, 24(18), 1663-1664.
5. Kolbezen, M. J., Metcalf, R. L., & Fukuto, T. R (1954). Insecticide structure and activity, insecticidal activity of carbamate cholinesterase inhibitors. Journal of Agricultural and Food Chemistry, 2(17), 864-870.
6. Miao Y, He N and Zhu J J (2010). History and new developments of assays for cholinesterase activity and inhibition. Chem Rev. 110: 5216 – 5234.
Reflective statement
We were formed in group of 4 people to conduct this experiment and research. In my group there was Lydia Chin, James Healy, Vincent Lee and myself: Shikha Surana. To start of with we exchanged our numbers and contact details so it would be easier to contact and discuss about this research. Once we finished the experiment we all decided to split our jobs for our group report. I did the introduction of this report, Vincent did the methods, James and Lydia did the results. We managed to finish and complete our task, but at times it was hard to contact people as there was no response. For my introduction I carried out research on by searching for keywords that were in the aim of the experiment. I started my research by google scholar, then pubmed and I couldn’t access the full text of those articles. Then I accessed the full text data of the articles that I specially wanted from usyd online database and read them thoroughly to get the full knowledge of this research, also how it is related to our findings and what is already known about it. Then I wrote dot points as I was reading all the journal articles, once I had gone through all of them by using the dot points I formed a draft of my introduction and then edited it until I thought it wasn’t okay and I tried to do it before our last group meeting, so we could review it and I could get some feedback from other group members and I can finalise my introduction. After group meeting there were couple of errors which I needed to fix for the introduction and after that introduction was done. We also reviewed results and method section which were undertaken by other group members and gave each other feedback on how to make it better. Other times when we couldn’t meet we usually contacted each other via facebook. That was the group work. For the discussion I used the same journals articles which I used in the introduction and worked through my discussion, edited and did referencing at last. Then I sent all my work to James as he is going to get everyone’s work and put it all in on document and submit it. Overall, it was really interesting and I enjoyed this research.
