Report Example: Calculations on Enzyme Activity

2021-07-05 12:01:57
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Harvey Mudd College
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Having the absorbance (at 420) and the reaction time, it is possible to calculate enzyme activity. However, this may not be possible as you need to have the extinction coefficient (epsilon:e) of the product obtained and then apply the Beer Lambert Abs = e c l ( l refers to the path length. For instance if you use cuvette of 1cm then you can possibly carry out a calculation of the concentration of the product that appeared or either the substrate that disappeared by Abs/el (Martano et al., 2016).

While calculating, it is prudent to be exclusively be careful with the units of e so as to determine C (often in mM). For example if you have C in mM and you are using 1ml as observed in the experiment you will have to assert that if c = 0. 326 nM in 1ml. then if the delta Abs is 1 minute then it means that it will be 0.326nM per 1min and you will have to know the quantity of enzyme put in the curvet for example using the data obtained in tube 2; 0.32nM then the catalytic constant (kcat) will be 100min-1.

If you have to calculate the enzyme activity as nmol/min/mg ( then it is a must to pinpoint the amount you put in the cuvette for example 0.2 mg in 1ml then it means you have 200nmol/min for 0.326mg you multiply by 100 to get 32.6nmol/min/mg in tube 1 and in tube 2 it will be 0.328 x 100 = 32.8nmol/min/mg which will be the enzyme activity.

Discussion

Ribose-5-phosphate isomerase (Rpi) serves as an enzyme used to catalyse the conversion of ribose-5-phosphate to riboluse-5-phospahte. Typically, it is a member or larger class of the isomerase components whose primary function is to catalyse inter-conversion of chemical isomers. In this experiment, the Rpi played a vital role in the biological metabolism in both the Calvin cycle and the pentose phosphate pathway (Klaassen et al., 2017).

tris (hydroxymethyl) aminomethane is the most commonly used biological buffer in most experiments involving the extraction of plant crude extracts. Silver beet is pH sensitive. During the process of centrifuging or precipitation and removal of on unwanted components of the extract, there is application of tris to ensure that a relatively stable PH is maintained. Broadly, the pH of on any extract could be influenced or can influence a number of cellular activities. Therefore, it is critical that a stable PH is maintained to ensure essentiality of the biological experiments (Klaassen et al., 2017). From the experiment, it was observed that tris is an effective biological buffer. It was possible to maintain a pH that was stable enough despite the several factors that would have changed the PH. However, it should not be forgotten that tris is sensitive to temperature and it should be used in its initial temperature PHed to ensure that accuracy is maintained.

Regarding the values obtained from the experiment, there were some minimal variations as compared to the conventional results of the same experiments performed. Based on the observations made, the variations is speculated to have resulted due to the following: Lack of accuracy in measuring of the reagents, temperature differences there was slight temperature variations that is expected to have influenced the results (Saha et al., 2016). Furthermore, other assumption for the differences of the data was due to poor or inaccurate recording of data, inaccurate timing as well as poor preparation of the experiment and the use of defective or faulty apparatus.

In any experiment conducted, there are typical errors that may arise due to lack of accuracy. Therefore, the best way to reduce such accuracy errors is to employ the use of several experiment while testing the same aspect. This was the reason for extending the experiment to experiment 4, and 5. Comparing the results, it was possible to come up with an accurate conclusion of the observations.

Additionally, while carrying out an experiment it is always critical that a control experiment is used. Purportedly, the use of a control experiment is necessary in order to display vital distinctive difference between the results obtained or observed and the practical aspect when the experiment is left blank without addition of any enzymes or the use of boiled enzyme essentially displaying similar characteristics. Therefore, in this experiment tubes 6 and 7 served purposes of control experiments (Klaassen et al., 2017).

In tube 3 the enzyme used was boiled, this process denatured the enzyme. An enzyme that has been denatured does not react or favours limited reaction. Therefore, there was no observable reaction in the tube where boiled enzyme was used. Is pentose-5-phosphate the only enzyme present in the final extract of silver beet? Yes, based on the experiment carried out, it was observed that pentose-5-phosphate is the only enzyme present in the final extract (Jin et al., 2017). This assumption was observed because any other enzyme tested was found negative but tested positive for pentose-5-phosphate.

Conclusion

Enzyme pentose-5-phosphate isomerase is crucial in both the Calvin (photosynthesis) and the pentose pathway. The most important compounds in this process are ribose-5-phosphate and ribose-5-phosphate in which the first compound is converted to the later in the presence of the enzyme (Pentose-5-isomerase). Conducting an experiment of the activity of the enzyme in the process is can be varied and will produce evidence of the function of the enzyme.

 

Reference

Jin, K., Li, L., Sun, X., Xu, Q., Song, S., Shen, Y., & Deng, X. (2017). Mycoepoxydiene suppresses HeLa cell growth by inhibiting glycolysis and the pentose phosphate pathway. Applied microbiology and biotechnology, 101(10), 4201-4213.

Klaassen, P., Van Suylekom, G. P., Gielesen, B. E. M., Broers, N. J., Wiedemann, B., & De Laat, W. T. A. M. (2017). U.S. Patent No. 9,551,015. Washington, DC: U.S. Patent and Trademark Office.

Klaassen, P., Van Suylekom, G. P., Gielesen, B. E. M., Broers, N. J., Wiedemann, B., & De Laat, W. T. A. M. (2017). U.S. Patent Application No. 15/407,341.

Martano, G., Murru, L., Moretto, E., Gerosa, L., Garrone, G., Krogh, V., & Passafaro, M. (2016). Biosynthesis of glycerol phosphate is associated with long-term potentiation in hippocampal neurons. Metabolomics, 12(8), 133.

Saha, R., Liu, D., Hoynes-OConnor, A., Liberton, M., Yu, J., Bhattacharyya-Pakrasi, M., ... & Pakrasi, H. B. (2016). Diurnal regulation of cellular processes in the cyanobacterium Synechocystis sp. strain PCC 6803: Insights from transcriptomic, fluxomic, and physiological analyses. MBio, 7(3), e00464-16.

 

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