Below is a balanced chemical equation of the ammonification of urea, and in the appendix 1.2 is a diagram of the reaction and how the enzyme urease functions.
The hydrolysis of urea is instantaneous important as it releases ammonia and carbon dioxide. Carbon dioxide is important for the photosynthesis of plants, which supplies oxygen back into the environment. Ammonia is essential as it is a main source of Nitrogen for vegetation. Nitrogen provides energy, protein and nutrients for vegetation and therefore influences how plants take form and function within. Urea is slightly acidic but when it reacts with urease to produce ammonia, it becomes basic. Because this occurs, phenolphthalein indicator C₂₀H₁₄O₄ is used to indicate this occurrence, as in an acidic solution it is clear, but turns bright pink when the solution becomes basic.
According to research urease can still function at a minimal temperature of 20˚C and between 10˚C and 30˚C the rate of reaction doubles. However the theories which will be evaluated is theory A, B and C. Theory A is based on a previous experiment which demonstrated that the optimum temperature of urease was 50˚C, however Theory B opposes this finding, stating that urease activity does not stop increasing until a temperature of 71˚C, thereafter, temperatures greater than 71 ˚C would denature the enzyme. Theory C states that the optimal pH of urease is approximately 7; therefore meaning that urease would function best in soils which were neutral; neither acidic nor basic.
The objective of this experiment is to support or oppose these theories by conducting several experiments, demonstrating urease activity, and the reaction time over a range of pH and temperatures. However the hypothesis is that the results will be in support of theory A and C, and in opposition to theory B.
Apparatus:
- Urea Solid
- Urease
- Phenolphthalein Solution
- 0.1M Hydrochloric Acid HCl
- 0.1M Sodium Hydroxide NaOH
- Distilled Water
- Two beakers
- Universal indicator paper
- Test tubes (and a control test tube)
- Test Tube rack
- Measuring cylinder
- 2mL Pipettes
- Stop watch
- Electronic Scale
- Water Bath
Procedure:
The first experiment was conducted in order to demonstrate the effects of temperature on the enzyme urease and its activity. Temperatures of 26˚C (room temperature), 37.5˚C, 50˚C, 60˚C, 70˚C and 75˚C were trialled separately, using a water bath. Each temperature was trialled ten times so that an average result could be obtained. Firstly the experiment was trialled at 26 ˚C (room temperature). In order to be organised during the experiment, the apparatus was set up and the materials prepared. To begin with, 0.2g of urea, measured on an electronic scale was dissolved throughout 60mls of distilled water in a beaker, and 2mls of this solution was then distributed into each test tube with the use of a 2ml pipette. Two drops of phenolphthalein solution was added to every test tube. Next, 2mls of urease was combined with the urea solution in a control test tube, as a point of reference. After the preparation of the apparatus and the materials, the experiment was conducted, combining 2mls of urease into each test tube, at separate intervals, in order to time each reaction. The reaction was timed as soon as the urease was dispersed onto the urea solution; as the reaction between urease and urea occurred instantaneously. The timing was ceased in reference to the control test tube, and the results were recorded. This experiment was conducted again for temperatures of 37.5˚C, 50˚C, 60˚C, 70˚C and 75˚C, using a water bath. The urease and test tubes were put in the water bath for these experiments.
Secondly experiments were carried out to investigate the result pH had on urease and its activity. A pH range of 4, 5, 6, 7 and 8 were trialled in order to demonstrate a diverse range of outcomes. Each pH was trialled four times. Firstly, a ph of 4 was trialled. The experiment was first organised by preparing the apparatus and materials. The urease was put into a water bath of 50˚C so that the reaction would occur quite rapidly. Using 0.1M of hydrochloric acid HCl, 20mls of distilled water in a beaker was carefully made acidic, measuring the pH with universal indicator paper. After a pH of 4 was achieved, 2mls of the acid solution was dispersed into every test tube using a 2ml pipette, and then 2 drops of phenolphthalein indicator was added in each test tube. A urea solution was then prepared. 0.5 g of urea was measured on the electronic scale and dissolved throughout 100mls of distilled water in a beaker. Using a measuring cylinder, 1ml of the urea solution was measured. Then using a pipette, 2mls of urease was extracted out of the 50˚C water bath and dispersed into a control test tube. The 1ml of urease solution in the measuring cylinder was then quickly poured into the test tube, so that the urease solution did not lose any heat. The experiment was then conducted dispersing 2ml of 50˚C urease into the prepared test tubes and adding 1ml of urea solution, trialling and timing four trials of the same pH in reference to the control. Experiments involving the pH of 5, 6, 7 and 8 were trialled using the same method, however 0.1M Sodium Hydroxide NaOH was utilised in order to make the solutions more basic.
In order to conduct both experiments, safety issues had to be carefully considered. Extra caution was taken when handling chemicals such as phenolphthalein, hydrochloric acid and sodium hydroxide. Both Hydrochloric acid and sodium hydroxide was handed carefully as they were extremely corrosive, and a danger to the skin and eyes, and additionally the vapours were extremely toxic. Sodium Hydroxide was also extremely corrosive and it was necessary to prevent contact with the skin and eyes. Phenolphthalein caused skin irritation and was harmful to inhale. Therefore the experiments were conducted in a laboratory, with direct access to sinks and running water. At all times a lab coat, safety glasses and closed in footwear was worn during the preparation and duration of the experiment. Windows were opened in order to ensure ventilation. Glass ware was also treated with caution.
Photos of the conducted experiment can be seen in the appendix.
Results:
Experiment 1Temperature:
2mls of urease was added to urea solution (0.2g of urea to 60mls of distilled water) at a range of different temperatures, and the reaction time recorded.
Observations:
After a few seconds the solution would begin to have a light pink tinge which would gradually become darker until it reached a baby pink colour.
TABLE: Reaction time(s) for all trials of temperatures experimented.
Experiment 2 pH:
2mls of 50˚C urease was added to solutions of ranging pH, and urea solution (0.5g of urea to 60mls of distilled water) was added. The reaction time was recorded.
Observations:
After a few seconds the solution would begin to have a light pink tinge which would gradually become darker until it reached a baby pink colour. However when a pH of 8 was tested when the phenolphthalein indicator was added, the solution automatically became pink but automatically became clear when the urease was added.
TABLE: Reaction time(s) for all trials of pH experimented.
Analysis:
Average time taken for urease to hydrolyse urea for a variety of different temperatures.
GRAPH: Summary of temperature results: average reaction times of urease for the temperatures experimented.
Average time taken for urease to hydrolyse urea for a variety of different pH
*urease at 50˚C
GRAPH: Summary of pH results: average reaction times of urease at pH 4 to 8.
Discussion:
The experiment was modelled in order to support or oppose several theories, and to demonstrate the importance of temperature and pH on enzyme activity. Theory A is based on an experiment which was previously conducted, which concluded that the optimum temperature of urease was 50 ˚C. However another source of research, theory B, contradicted theory A, and stated that the rate of reaction for the enzyme urease did not stop at 50 ˚C, but in fact continued increasing until 71 ˚C. With an intention to investigate these theories further, experiments were conducted at temperatures below 50 ˚C, 50 ˚C and above 50 ˚C.
Initially temperatures of 26 ˚C, 37.5 ˚C, 50 ˚C and 60 ˚C were chosen to first examine theory A. If at the chosen temperatures of 50˚C and below, urease activity had not declined, then both theory A and B would still be feasible until opposed by contradicting results. A trial at 60˚C would then take place in order to determine whether the enzyme activity had decreased. If enzyme activity had decreased at the temperature of 60˚C theory B would no longer be feasible. However if at 60˚C enzyme activity had not decreased but had conversely increased, theory B would still be possible, but the results would be against theory A. Further trials at 70˚C would then take place, and if urease had not yet denatured, experiments at 75˚C would then be conducted. If at 75˚C enzyme activity had increased yet again both theories would not have been supported by the conducted experiment.
The data collected from experiment 1, showed that between temperatures of 26 ˚C and 50 ˚C there was an increase in rate of reaction. This is clearly seen as at 26 ˚C the reaction time for urease to hydrolyse urea was 82.92s compared to 50 ˚C where the reaction time was 54.93s, a decrease of 33.76%. Additionally, the graph provides a clear visual representation of this data, and as a result the decline in reaction time is displayed suitably and easily interpreted. This is a definite indication and demonstration of the relationship between temperature and the time taken for an enzyme reaction to occur. This data clearly shows that as temperature increases, the rate of enzyme activity also increases. When the experiment was trialled at 60 ˚C in order to determine whether theory A was irrelevant or relevant, unexpected results were obtained. At 60 ˚C, urease denaturation and a decline in enzyme activity was expected, however urease activity had in fact increased, and the average reaction time was 45.63s. Since the enzyme had not denatured after 50˚C, theory A was invalid, and therefore further experiments were conducted to trial theory B’s plausibility.
In order to determine whether theory B could be applied to this experiment’s investigation, it was necessary to trial further temperatures above 60˚C. Temperatures of 70 ˚C and 75 ˚C were chosen to trial theory B, and when conducted, the results from the experiment showed that theory B was feasible. At 70˚C the average reaction time was 41.27s, a 50.23% decrease from the average reaction time at 26˚C. There was a dramatic decrease of 166.13% in enzyme activity at 75˚C, the average reaction time resulting in 109.83s. This also demonstrates that enzymes have an optimum temperature and that when this temperature is exceeded, enzyme activity decreases due to enzyme denaturation.
From the data it was concluded that between the temperatures of 26˚C to 50˚C urease enzyme activity increased at a higher rate compared to 50˚C to 70˚C. The difference in average reaction time between 26˚C and 50˚C was a decrease of 27.9s, however, there was only a decrease of 13.16s between 50˚C and 70˚C. This shows that the rate at which enzyme activity changes, increases rapidly over a certain amount of time but then gradually decreases until the optimum temperature is reached. According to the experiment conducted the optimum temperature is likely to be approximately 70˚C to 74˚C, but there is a possibility of denaturation anywhere between 71˚C and 75˚C. The difficulty of this experiment is that it does not actually reveal the optimum temperature and the exact point of denaturation and therefore does not fully ‘support’ theory B. The results from experiment 1 simply imply that there is a high possibility that theory B is plausible. In order to be accurate and have a definite result of the optimum temperature and denaturation temperature, more temperatures would need to be investigated. The results of the conducted experiment merely give an outline to possible temperatures which may be the optimum or denaturation point. Therefore, because theory B wholly suggests the optimum and denaturation points, this experiment cannot completely support theory B. As the temperatures which were trialled were not periodical, a curve joining the points of the graph was not drawn.
Theory C is associated with the optimal pH environment of urease, and is specified to be approximately a pH of 7. Hence in order to support or oppose theory C, it was necessary to trial urease activity in acidic, neutral and basic conditions. If urease activity denatured before a pH of 7 was achieved, or did not denature after a pH of 7, the experiment would have opposed theory C. However if enzyme activity increased from an acidic solution to a neutral solution, and then began to decrease and denature in a basic solution, theory C would be supported. In order to conduct this experiment a pH of 4, 5, 6, 7 and 8 were chosen in order to show an increase in enzyme activity towards the optimum pH and then a decrease as the enzyme denatures.
A clear increase in urease activity was demonstrated within the pH environments of 4 to 7. The average reaction time of urease at an environment of pH 4 was 107.48s, and at a pH of 7 the average reaction time was 49.17s, a significant decrease of 54.25%. These results reveal that urease does not function well in an acidic environment and therefore the time it takes to hydrolyse urea is longer. However when the urease was put into a basic solution of pH8, the enzyme activity decreased and the enzyme began to denature. The average reaction time at a pH of 8 was 79.48s, a 38.14% increase from the reaction time at a pH of 7. Therefore from the experiment results, and as seen on the graph, it would be correct to conclude that the optimum pH conditions for urease would be approximately 7 or neutral. As a result, these experiment findings highly support theory C, but similarly to experiment 1 does not accurately show the optimum or denaturation point associated with pH. A line connecting the points on the graph was drawn, because the pH tested was periodical, and therefore more accurate assumptions could be made.
Both experiments 1 and 2 would need to be improved in order to achieve more accurate results, as it was likely many errors and inconsistencies were made. In both experiments, a judgement of sight was heavily relied on, in order to determine whether the reaction had finished occurring. When the solution in the test tube had finished turning pink, it was assumed that the reaction had also finished. The pink colour was a result of phenolphthalein, which was clear in an acidic solution and pink in a basic pH above 8. In this experiment, the solution turned pink because ammonia (which is basic) was being produced. However, a judgement of sight is inefficient as it is not extremely reliable, as there may be difficulties in differentiating the colours between the trial test tube and the control. Similarly it was difficult to distinguish when the solution had actually finished turning pink, as when the experiments were trialled, it was found to be that not all the solutions transformed into the same shade of pink compared with the control and the other test tubes. This may be caused by a slight difference in pH. Working in partners may have also increased accuracy, as there would have been a second opinion judging the colour change. Additionally, the concentration of urea in the solutions prepared, differed in both experiment 1 and 2. Experiment 1’s solution had a molarity of 0.0476M, having a concentration of 1g of urea to 300ml of distilled water (1:300), and experiment 2’s solution had a molarity of 0.0714M, a concentration of 1g of urea to 200ml of water (1:200). In order to improve consistency and increase the comparability of the results, the same concentration of urea should have been applied to both experiments.
When experiment 1 was conducted, different temperatures were trialled using a water bath. The difficulty of using a water bath is that heat is constantly being lost to the air, and therefore the temperature is constantly changing. For an experiment on enzyme activity, every temperature trialled is important, and consistency is crucial in order to find the optimum point. For example, when urease activity was trialled at 70˚C the temperature constantly varied between 67˚C to 70˚C, hence the data collected was not precise. Another inconsistency was that some of the test tubes, and enzyme activity was absentmindedly interfered with. During the reaction some of the test tubes were swirled and shaken so that the urease enzyme solution would be distributed into the urea solution. However it was not realised at that point that in doing so, the rate of reaction was being increased, as the shaking and swirling of the test tubes multiplied rapid molecule movement, hence increasing the chance of urea and urease active site interaction. These errors only affected temperatures trialled at 26 ˚C to 50 ˚C, and did not affect the results of the temperatures at 60 ˚C to 75 ˚C, as this inconsistency was soon realised before those trials were conducted. When this experiment is next conducted, temperatures which are periodical (ex. 5 ˚C, 10 ˚C, 15 ˚C, 20 ˚C….) should be trialled in order to come to more accurate assumptions and conclusions.
The main problem when conducting experiment 2 was creating solutions of different pH levels using hydrochloric acid HCl and sodium hydroxide NaOH. Universal indicator paper was used to check the pH of the prepared solution, and judgement of sight was again used to determine the pH of the solution. There were often difficulties in reading the pH, and other people’s opinions were used to determine the pH. It also has to be taken into account that the pH scale is not discrete and it is possible to have a pH of 4.3 or 5.8. However with the utilised equipment, it was impossible to fully distinguish an accurate pH, and therefore the pH trialled during experiment 2 were simply approximations and estimates which, if measured by proper equipment, would be inaccurate. Consequently the results are also inaccurate; when analysing the graph it would be most likely that a person would say that the optimum pH for urease is pH7, but because the pH trialled were just approximations and there are pH which were not trialled, this result would also be an approximation. From the results it could be said that the optimum pH for urease is approximately between a pH of 7 to 7.9, and that the denaturation point could approximately be a pH of 7.1 to 8.
Evaluation:
This experiment was conducted in order to investigate the importance of the relationship between enzyme activity and variables such as temperature and pH. There were also several theories, A, B and C which were investigated during this experiment. Theory A was based on a previously conducted experiment which suggested that the optimum temperature of urease was 50˚C, contrarily theory B stated that enzyme activity increased until a temperature of 71˚C. Theory C established that the optimum pH of urease was approximately 7. From the gathered results which were carefully analysed, it was found that theory B and C was supported by the conducted experiment. Furthermore, an obvious relationship between enzyme activity and the temperature and pH of an environment, was demonstrated by the results and could be visually analysed on the graphs. Primarily this experiment was a success, in the perspective that it supported the plausibility of several theories; however, many improvements and adjustments could have been made to overall improve the experimental method and results.
If the experiment was to be conducted again several adjustments would need to be made to improve the consistency of this experiment and to achieve more accurate results. Improvements such as using solutions with the same concentration of urea, ensuring that the test tube was not shaken or swirled to prevent interfering with the reaction, preventing heat loss from the water bath and using an electronic pH meter to test for pH. This experiment would also be more accurate if two people were working together as there would be two opinions judging the reaction. Other variables which could have also been tested are the effects of substrate and enzyme concentration, the amount of carbon dioxide produced, and the effects of certain chemicals on the rate of enzyme activity.
Conclusion:
In conclusion, the hypothesis was both supported and opposed by the results of the overall experiments. The hypothesis theorised that the optimum temperature of urease was 50˚C; however the results of this experiment suggested that the optimum temperature was approximately 70˚C. The results of this experiment implied that the optimum pH of urease is approximately 7, which supports the theory from the hypothesis. The experiment was overall successful in demonstrating the relationship and effects of variables such as temperature and pH towards an enzyme.
Appendix
The Experiment
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