Biochemistry experiment 4A and 4B – Time course of an enzyme catalysed reaction/ Effect of pH on the rate of an enzyme catalysed reaction

  • Introduction

Enzymes are organic molecules comprising of proteins. They are highly specific in the way in which each one catalyses a unique individual reaction. As enzymes are comprised of proteins they can denature if not kept in idyllic conditions, therefore they lose their function thus stopping any reactions (Gomes and Rocha-Santos, 2018). As enzymes are highly specific they each work differently in varying degrees of pH. (Krogdahl et al., 2015) Enzyme catalysed reaction experiments can be extremely important and beneficial, especially in some fields of medical research. Tapper et al., (2017) suggests that elevated liver enzymes within a patient can indicate detection of a wide variety of liver diseases. With the use of enzyme catalysed reaction experiments, several different tests to determine which type of disease is present can be done all at once. This is a more efficient and affordable way to obtain fast results.

 

  • Materials and methods

 

2.1 Part A: Time course of an enzyme catalysed reaction

5 sets of 3 microcentrifuge tubes were firstly labelled A1-E3. The following solutions were then added to each microcentrifuge tube; 0.2 mL distilled water, 0.25 mL sodium acetate buffer solution (pH5.0) and 0.25mL para-nitrophenol phosphate (pNPP) solution. Each tube was then left to equilibrate at room temperature for 5 minutes, then immediately transferred to a water bath of 30  where they were left for a further 5 minutes. 0.05 ml of enzyme was then added to each tube and gently agitated. In order to maintain accurate timings, the enzyme was added to each tube at 1-minute intervals. The times were recorded by a clock timer. The 5 sets of microcentrifuge tubes were incubated for 5, 10, 15, 20 and 30 minutes (i.e. tube sets A1-A3 incubated for 5 minutes, tube sets B1-B3 incubated for 10 minutes etc). At the end of each incubation period the reaction was stopped by adding 0.5 ml sodium carbonate and the tube was then placed back into the test tube rack. Before determining the absorbance of each reaction mixture, a blank solution was prepared by adding the following solutions into a microcentrifuge tube; 0.2 mL water, 0.25 mL sodium acetate buffer, 0.25 mL pNPP, 0.5 mL sodium carbonate and lastly 0.05 mL of enzyme. To determine the rate of absorption the spectrophotometer had to firstly be set to calibrated with the blank solution. The absorbance of each reaction mixture was measured at a wavelength of 410nm. Before the absorbance could be measured by the spectrophotometer, the contents of each microcentrifuge tube were transferred into a low-volume cuvette.

2.2 Part B: Effect of pH on the rate of an enzyme catalysed reaction

The same basic procedures were followed as mentioned before in section 2.1. Although, for this experiment 4 sets of 3 microcentrifuge tubes were used, with each set containing a different pH buffer. There were 4 different pH buffers provided which included; pH 3, pH 5, pH 7 and pH 9. Each of the 4 sets of microcentrifuge tubes were all placed in a water bath at 30  for 10 minutes. Following completion of incubation, the reaction was then stopped by adding 0.5 mL sodium carbonate to each tube.  The incubation period was chosen as 10 minutes sat in the centre of the linear trend line and this was deemed the most accurate choice. The blank used to calibrate the spectrophotometer in part B was prepared in the exact same way as the blank that was used in part A. After calibration of the spectrophotometer, the absorbance of each reaction mixture was again measured at a wavelength of 410nm. The raw data obtained from the spectrophotometer readings was then converted into the amount of pNP produced within 10 minutes. This data was obtained by using equation 1. Equation 2 was then used to convert the data obtained from equation 1 into the rate of pNP produced per minute. A mean average was then taken from this final set of data to be used to plot the graph that is shown in figure 3.

Equation 1 – pNP produced (μmol)= (absorbance @ 410nm-0.0035)/3.1576

 

Equation 2 – rate of reaction (μmol / min)=(amount of pNP (µmol))/(10 (min))

 

  • Results

To begin the results, the pNP calibration data provided in experiment 4A was converted into a graph as shown by figure 1. The equation provided by this first graph was then used to convert the absorbance values that were obtained in experiment 4A into concentration of pNP produced (µmol).

Figure 1

 

Figure 1 – pNP calibration data plotted to obtain equation y = 3.1576x + 0.0035.

This equation was then used to convert the recorded 3 absorption values for each set of 5 into µmol of pNP produced for each different time frame (5 minutes, 10 minutes etc.). A mean average was then taken from the 3 absorbances samples from each set. This mean average was then plotted on the graph shown in figure 2. The graph was used to calculate an accurate incubation time for the samples used in experiment 4B.

Figure 2

 

Figure 2 – Mean averages taken from the three samples recorded for the 5 different sets, plotted against the time each set was incubated for. The standard deviation from all the results recorded for each of the 5 sets was calculated to be used in the form of error bars to indicate accuracy.

It was decided that 10 minutes would be the optimum incubation time for the samples produced in experiment 4B. This decision was made as 10 minutes sits in-between the linear phase. This was deemed appropriate as 5 or 15 minutes may not have been as accurate as the sat at the very beginning and the very end of the linear phase.

Figure 3

Figure 3 –  The rate in which pNP (µmol) is being produced per minute at 4 different levels of pH, including; pH3, pH5, pH7 and pH9. The samples were incubated for an optimum time of 10 minutes in a water bath at 30.

 

  • Discussion

Acid phosphatase hydrolyses phosphate from a variety of molecules which contain a phosphate group (Wang and Liu, 2018). The substrate used within this experiment was para-nitrophenol phosphate (pNPP). When subjected to acid phosphatase, pNPP is hydrolysed to create para-nitrophenol (pNP). Within an alkaline solution pNP can be detected as it turns yellow in colouration (Jez et al., 2016). This is beneficial as the colouration can be assessed with the use of a spectrophotometer to monitor the rate of production of pNP. Production rate of any enzymes relies on specific environments.

Observing figure 3 it is clear to see that the idyllic pH for acid phosphatase to produce pNP is pH 5. As enzymes are acidic in nature it could be assumed that pH 5 would be optimum as acid phosphatase is clearly acidic in nature so would work best in an acidic environment. pH 5 is deemed acidic without it being too harsh that it becomes destructive to the enzyme. It could also be argued that pH 7 would be ineffective as the solution would still be too neutral for a productive enzyme reaction rate. This argument can be supported by Behera et al., (2017) who also carried out an enzyme assay on acid phosphatase. It is stated within this experiment that the optimum acid phosphatase activity was recorded at a temperature of 48 at a pH of 5. However, although the optimum pH for both experiments was deemed pH 5, two different temperatures were used which would have affected both results. Nevertheless, both arguments can be debated as Jennifer et al.,(2015) suggests that the optimum environment for the production of enzymes is pH 9 at a temperature of 40. The comparison of these arguments could propose that further study should be completed to investigate the effects of varying temperatures on the production of enzymes.

Optimal conditions are essential for enzyme production. A change in environment can cause an enzyme to denature, thus meaning they stop functioning, which will result in a stop of production. (Saoudi et al., 2017) Denaturation is the process in which an enzyme will undergo threatening and even destroying structural change. Although enzymes are subject to structural damage, no reaction would be strong enough to break the peptide bonds between amino acids, so the enzymes primary structure would remain unchanged (Cheng et al., 2015). As the process of denaturation causes the enzyme to change structure, this then means that the specific substrate matched with that enzyme can no longer bind as the active sight region has been impaired resulting in the substrate and enzyme no longer being a match (Witkowska et al., 2018).

 

  • Conclusion

To conclude, the production rate of pNP was measured over numerous lengths of time to investigate the most productive time span for pNP production. This information was then used to evaluate the best incubation time for the solutions used with varying degrees of pH. The experiment deemed that pH 5 was the optimum environment for pNP production. Literature suggests that this is due to the enzyme working well within an acidic environment because it itself is acidic in structure, although the pH concentration wasn’t too acidic that it would cause any structural damage to the enzyme, causing it to denature.

 

 

 

 

References

Behera, B. C., Yadav, H., Singh, S. K., Mishra, R. R., Sethi, B. K., Dutta, S. K. and Thatoi, H. N. (2017) ‘Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India.’ Journal of Genetic Engineering and Biotechnology, 15(1) pp. 169–178.

Cheng, B., Wu, S., Liu, S., Rodriguez-Aliaga, P., Yu, J. and Cui, S. (2015) ‘Protein denaturation at a single-molecule level: the effect of nonpolar environments and its implications on the unfolding mechanism by proteases.’ Nanoscale, 7(7) pp. 2970–2977.

Gomes, A. R. and Rocha-Santos, T. A. P. (2018) ‘Enzyme Assays☆.’ In Reference Module in Chemistry, Molecular Sciences and Chemical Engineering. Elsevier.

Jennifer, E. B. M., Sathishkumar, R. and Ananthan, G. (2015) ‘Screening of extracellular hydrolytic enzymes production by ascidians (Polyclinum glabrum, Microcosmus exasperates and Phallusia arabica) associated bacteria from Tuticorin, Southeast coast of India.’

Jez, J. M., Ravilious, G. E. and Herrmann, J. (2016) ‘Structural biology and regulation of the plant sulfation pathway.’ Chemico-Biological Interactions. (Special Issue on Sulfation Pathways), 259, November, pp. 31–38.

Krogdahl, Å., Sundby, A. and Holm, H. (2015) ‘Characteristics of digestive processes in Atlantic salmon (Salmo salar). Enzyme pH optima, chyme pH, and enzyme activities.’ Aquaculture. (Proceedings of the 16th International Symposium on Fish Nutrition and Feeding), 449, December, pp. 27–36.

Saoudi, O., Ghaouar, N., Ben Salah, S. and Othman, T. (2017) ‘Denaturation process of laccase in various media by refractive index measurements.’ Biochemistry and Biophysics Reports, 11, September, pp. 19–26.

Tapper, E. B., Saini, S. D. and Sengupta, N. (2017) ‘Extensive testing or focused testing of patients with elevated liver enzymes.’ Journal of Hepatology, 66(2) pp. 313–319.

Wang, L. and Liu, D. (2018) ‘Functions and regulation of phosphate starvation-induced secreted acid phosphatases in higher plants.’ Plant Science, 271, June, pp. 108–116.

Witkowska, D., Cox, H. L., Hall, T. C., Wildsmith, G. C., Machin, D. C. and Webb, M. E. (2018) ‘Analysis of substrate binding in individual active sites of bifunctional human ATIC.’ Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics, 1866(2) pp. 254–263.

 

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