The background information about enzymes has been taken and edited from Microsoft Encarta 95.
This background knowledge emphasises the significance of enzymes to humans and how they help us in many ways.
PREDICTION:
I predict that when testing the specificity of the enzyme, the more similar the substrate is used the easier it would be to be broken down. This is because the rate of reaction would be quicker, due to the urease being able to break down the substrate quicker, therefore creating a bigger surface area for the urease enzyme to work on. The stronger the substrate is, the slower the rate of reaction would be.
I also predict that as the temperature increases the rate of reaction would increase, this is because the particles are more vibrant. Due to the rise in temperature, the heat energy transfers to the enzymes as kinetic energy. This energy is shown by the enzymes moving considerably fast, resulting in more successful collisions, causing the substrate to be broken down quicker. The initial energy transferred to the particles is known as the ‘activation energy’, this is needed to break the bonds, to break down the substrate. However, as my background knowledge mentions, the enzyme would soon denature after a certain temperature. I believe the optimum temperature would occur around 40ºC, after this optimum temperature the rate of reaction would decrease due to the urease enzyme denaturing.
The reactions to this equation are:
UREA SOLUTION
+ H2O 2NH3 + CO2
urea + water ammonia + carbon dioxide
The urea bond is formed NH2, which are two amino groups.
METHYLUREA SOLUTION ETHANAMIDE SOLUTION
NH2 NH2
CH3 – C 0 = C
NH2 CH3
The rest of the equation is the same after the substrate.
APPARATUS:
- 0.25M urea solution - used to test specificity of the enzyme.
- 0.01M hydrochloric acid – to turn the indicator a slight red colour.
- 1% urease solution – the enzyme being used.
- Universal Indicator – to test whether acid or alkaline.
- Methylurea – used to test specificity of the enzyme.
- Ethanamide - used to test specificity of the enzyme.
- Beakers – to pour different solutions into, and to act as a water bath.
- Bunsen burner – to heat, and change the variable.
- Tripod – to hold beaker up.
- Gauze – to hold beaker up.
- Boiling/ test tubes – to heat mixed solutions.
- Test tube rack – to hold boiling/test tubes.
- Stop-clock – to time how long the solution takes to turn alkaline.
- Measuring cylinder – to measure certain volumes of certain solutions.
METHOD:
The steps below show how the experiment of testing the specificity of the enzyme happens:
- To 5cm² of a 0.25M urea solution add five drops of Universal Indicator followed by a couple of drops of 0.01M hydrochloric acid until the indicator has just changed to a distinct red colour.
- Add 1cm² of 1% solution of urease active meal (which has been similarly treated with indicator and acid), and then note how quickly the pH of the solution changes by observing the colour. To measure how long it takes for the pH to changes, start the stop-clock as soon as the two solutions are mixed, and then stop it as soon as the colour is changed totally.
- Repeat the experiment with 0.25M solutions of compounds, which have structural similarities to urea and would therefore be able to be hydrolysed by urease to ammonia. Suitable compounds include ethanamide and methylurea.
- Boil 1cm² of the 1% solution of urease active meal for 30 seconds, and then cool to room temperature. Repeat the first experiment using the boiled urease solution.
- Then do this similar method using temperature as the variable.
Repeat each experiment 2 times each so an average of the results can be taken. By taking an average the results would be reliable and more accurate.
The steps below show the experiment of temperature affecting of the enzyme:
- I will begin by setting up water baths at 30°C, 40°C, 50°C 60°C and 70°C.
- I will first prepare the solution, which was made before in steps 1 and 2. However, before I add the urease, I would have putted the urease in the specific water bath for several minutes.
- Add the solution of urea and hydrochloric acid with the indicator and start the stop clock
- As soon as the solution has turned blue indicating alkaline, this will be done through observation, stop the stopclock.
- Record the time taken, and the final colour of the indicator. Use the time to calculate the rate of the reaction.
- Again repeat the method for the temperature three times to allow an average to be taken.
The temperature method only needs to be carried out with the substrate urea.
Below shows how my results tables will look before any values are entered.
Below is the results table showing the specificity of the enzymes.
Below is the results table showing the temperature being the variable affecting the enzyme, urease.
FAIR TEST:
Using the same equipment, pr similar equipment would help in making the experiment fairer, as by using the same equipment, the measurement would all be totally accurate. I would also try to prevent the contamination of the urea and urease jars, and acid jars. This also includes using the right pipettes for each solution. I will also make sure I do the experiment on my own, as this better, as I would stop and start the stop-clock, according to when my eyes see the right time or the right reading.
SAFETY:
I will behave at all times during the experiment, especially when holding, beakers, boiling tubes or pipettes due to them being glass objects. Therefore, I will be using a test tube rack to avoid breakages. I will also wear goggles when using the bunsen burner when testing the temperature variable.
DIAGRAM:
I WOULD HAVE DONE A DIAGRAM
RESULTS:
Below shows the results of the experiment carried out when investigating the specificity of the enzymes.
TABLE 1
The table on the following page shows the results of the experiment carried out when investigating the temperature being used as the variable affecting the enzymes.
In both tables, I have also recorded the final colours of the solutions after the enzyme has broken down the substrate fully. By looking at the layout of the table, you can notice how three sets of results were collecting, thus meaning three experiments were carried out. This was done so, so averages could be made, making the results more reliable.
On the separate sheets are two line graphs representing data on table 1. Graph 1 and graph 2 show lines of best fit for the average time and rate of reaction of each substrate used. There are also two graphs representing data of table 2 on a separate sheet.
TABLE 2
By looking at the results in table 1, you can see that as the substrate changes to methylurea and then to ethanamide, the rate of reaction decreases. These results prove my prediction was correct, as I mentioned in my prediction that the more the similar the substrate used to the enzyme, the quicker the substrate would break down creating a bigger surface area for the enzyme to work on, thus causing the reaction to work faster.
The average time taken, measured in seconds proves the quickness of certain reactions. The average time of urea, when reacting with the urease was 46.6 seconds. This dramatically increased when a more complex and un-similar substrate was used, in this case methylurea. The average time then increased to 124 seconds. The results also showed that ethanamide is an even more complex and difficult substrate, in which the urease enzyme finds it extremely hard to break down. This is because no average time, and therefore no rate of reaction could be determined due to the enzyme not being able to break the substrate down at all. The stop-clock ran over five minutes of timing, therefore I concluded that the enzyme could not break down this substrate. Graph 1 and graph 2 (on the separate sheet) present this data in a way that you can predict the readings of when urease reacts with ethanamide. The best line of fit can be used to do this.
By looking at the results in table 2, you can see that the results show the optimum rate of the enzyme to be working between 40ºC and 50ºC. The curve drawn on graph 3 shows that the optimum rate of the enzyme urease occurs at 43ºC. This value is very inaccurate as according to my background knowledge the optimum rate of an enzyme occurs at 37ºC. My experiment shows that urease is an enzyme that can withstand higher temperatures than other enzymes and work better at an even warmer temperature. However there is also the possibility that my experiment could have been slightly wrong in certain places, as specific mistakes made may have caused this optimum temperature to come about.
The results on table 2 also show how the urease enzyme works poorest at 30ºC. As at 30ºC the average time of when the enzyme broke down the 0.25M Urea solution mixed with the 0.01M Hydrochloric solution was 183.3 seconds. This reading dramatically decreased to 76.6 seconds, once the temperature was increased to 40ºC. The rate of reaction of the enzyme-increased further once the temperature was increased to 50ºC. As the average time decreased to 56.3 seconds, however at 60ºC, the average time increased more than double to 156.5 seconds. This clearly indicated that the urease enzyme was denaturing and was therefore causing the rate of reaction to decrease. Graph 3 and 4 show this clearly, as on graph 3, the curve increases steepness going upwards, graph 4 shows the curve steeply falling indicating a decrease in rate of reaction.
EVALUATION
I believe my experiment was a good way of investigating the aims, as this experiment wasn’t too time consuming, where it was extremely difficult to obtain results. Plenty of experiment could have been done, which were done allowing reliable and accurate average to be calculated. However, there was a problem with a substrate used in the experiment, ethanamide could not be broken down by the enzyme. This created a gap in the results in my experiment as ethanamide didn’t have a reading at all, as I had left the stop clock running after 5 minutes through the experiment with still no change of colour of the solution. The enzyme could not break down Ethanamide; therefore no result was entered in the table. I f I had repeated this experiment another time I would have used a substrate which was possible of being broken down no matter how long it takes for the enzyme to do so.
I have circled an anomaly on graph 3 and 4, which had occurred when I was investigating the rate of reaction of the enzyme at 40ºC. This anomaly may have occurred due to the water bath not keeping at 40ºC. During my experiment I tested the water baths to see if they all were at the right temperatures, I found they were not. I could do nothing about this, as other people experiment would have been unfair if I changed the temperature. Therefore, as a solution I would keep all temperatures at the right temperatures before I start the experiment. I would do this if I were doing the experiment again by myself so other peoples experiments could not be disturbed. There were also other factors affecting the results, which mainly involved human error. People working in my group may have failed to start and stop the stop clock at the right times. Other member of the class may have contaminated different solutions with each other, I was careful and avoided this but other people may have not been. People in my group forgot to leave the two solutions in some water to acclimatise, so each solution starts off with the same temperature. If this experiment was done extremely accurately, with many-repeated experiment creating a more accurate average, then a reading may have been made for ethanamide. Avoiding these factors and improving certain parts of the apparatus and experiment would have achieved then better results. Apparatus like the water baths caused the experiment to be unfair, as they were not constant at their specific temperatures.
Above all I believe this experiment went well, as I had fulfilled my objectives and collected a group of accurate data.
If I wanted to investigate the activity of enzymes further I could have used other variable, which would affect the enzymes and rate of reaction. Other variable that I could use could be, varying the pH levels and concentration of the solutions or using different amounts of a particular solution.
BIBLIOGRAPHY
Other resources that helped me during the investigation:
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Letts Biology Revision Guide