The substance that enzymes act on is the substrate. The substance formed by the reaction is the product and the site on which the enzyme takes place is called the active site.
The diagram below shows how the enzyme and substrate work together to form a product:
I will produce graphs to display my findings which will help me to analyse my results and draw suitable conclusions. I have prediction that the rate of reaction will increase as the concentration is increased. I also predict that the rate will be directly proportional to the concentration. Therefore, I will expect my results to form a straight line on my graph.
Apparatus
- Urea
- Urease
- Ethanoic acid 0.1m
- Universal indicator solution
- Water
- Test tubes
- Stop clock
- Syringe
- Pipette
- Measuring cylinder
- Glass beaker
- Thermometer
- pH chart
- Safety glasses
Method
- Using a pipette measure 5cm³ of urea and 2cm³ of ethanoic acid and put into a test tube.
- This will make the solution acidic so that when the universal indicator is added we can clearly see the changes the solution undertakes when the urease is added. (The ammonia it produces is alkaline so will turn the solution green-blue)
- Add 10 drops of universal indicator to the acid and urea in the test tube. The solution will turn a red-orange colour.
- Now add 3cm³ urease solution and start stop clock immediately as it is in contact with the urea and acid in the test tube. The solution will change colour from red, to yellow and finally green.
- Observe the test tube carefully, watching for the disappearance of the acid. This should be when the solution turns a green-blue colour, or pH 8.
- Stop the clock when the solution reaches pH 8.
- Record time and then repeat experiment 3 times and calculate the average total.
- Now repeat the process, changing the quantity of urease and adding the correct amount of water as is appropriate. Repeat until each concentration has been tested three times.
Fair Test
In order to keep this test fair and ensure my results are accurate and reliable, I must keep certain things the same. I will aim to keep the temperature the same throughout the experiment as this will affect the enzyme - if the temperature gets too hot, usually around 45-50°C, then the enzyme becomes denatured and cannot perform its function correctly. I can make sure this happens by checking the temperature with a thermometer. I will make sure there is always the same volume of solution in the test tube, as this could give different results if it is not the same each time. Also, the method of comparing pH I will use to determine the end of the experiment must be the same, so that all the results are fair. This will be monitored using universal indicator solution as I will need to know the pH to find out when the reaction has ended. I will stop the experiment once the solution reaches pH 8.
Obtaining Evidence
Analysis
After collecting my information, I originally drew graphs plotting the concentration of the urease against the time taken for the reaction to take place. However, these graphs did not show me the rate of reaction of the experiment, which is what I am investigating, so I calculated the rate of reaction using the equation: rate of reaction = 1
time
Using these results (see Obtaining Evidence) I was then able to draw graphs plotting rate of reaction against concentration of urease to show how the two are related.
As we can see from the graph for experiment 1, the rate of reaction increases as the concentration of the urease is increased. As the rate of reaction increases, it does so proportionally. The line of best fit is straight and there are no anomalous results plotted in this graph. Therefore, I can conclude that this experiment has worked well and the results appear to form the expected pattern.
The graph for experiment 2 has a similar pattern, however point 0.6 appears to be an anomalous result. Here, the rate of reaction appears to be far quicker than it should be. The temperature of the solution is 20°C and so this should not have affected the result, because although enzymes tend to work faster in warmer conditions, 20°C is not particularly warm. I suggest this result is anomalous because of human error or inaccuracy, as it is not very clear when the experiment has actually finished and the reaction has been completed. Therefore, some leeway must be allowed for slight inaccuracies, but the overall trend is still easily identifiable: the rate of reaction increases as the concentration increases.
The graph displaying the results for experiment 3 has an almost perfect line of best fit. The results are in proportion and they all seem to fit the pattern well. The rate of reaction increases more quickly between points 0.3° and 0.5, where there is a difference of 0.00094 between the rates. This rate then continues to increase but at a less steep rate as the difference between the rates of reaction of concentrations 0.5 and 0.6° is 0.00027. However, instead of continuing to rise at a less steep rate, the rate of reaction for point 0.83° then accelerates to 0.00123 faster than the rate of reaction for the concentration below it, which is 0.6°. The difference in rate then drops again to 0.00042 between 0.83° and 1. Despite these dramatic sounding results, the graph shows a clear line of best fit and it is evident that the experiment agrees with my prediction that rate increases with concentration.
Overall these results appear to be reliable and show a distinct pattern forming, but to increase the accuracy of my results, and therefore increase the accuracy of my conclusion, I have taken averages for each concentration from all three experiments and plotted them on a scatter diagram.
The line of best fit for this graph is almost perfect. All the points fit in the trend. There is positive correlation and the rate increases in proportion as the concentration of the urease increases. This proves that the evidence I have collected is reliable and allows me to conclude what I have found out in this experiment with a great deal of accuracy.
Conclusion
At the beginning of this investigation, I predicted that as the concentration of the urease becomes weaker, the time taken for the reaction to be completed would increase so that as the concentration is increased, the rate of reaction also increases. I also predicted that the rate of reaction would be directly proportional to the enzyme concentration. I thought this because as the enzyme concentration rises, the number of active sites that are available to react with the substrate also rises and so the rate of products being formed is increased.
This is because as the enzyme concentration rises, it makes more active sites available to interact with the substrate, thus raising the rate of products being formed. The same thing would happen if the amount of substrate was limited because this too would limit the rate of reaction. The enzyme can only work on one substrate at a time and so if there are more frequent numbers of substrate then they have to wait until the an enzyme molecule is free to react with it.
In order to understand my results properly, I will recap on the lock and key formula: The substance that enzymes act on is the substrate. In this experiment the enzyme is urease and the substrate is urea. The substance formed by the reaction is the product and the site on which the enzyme takes place is called the active site. The active site of the enzyme acts like a lock. It’s specific shape only allows the precise amount of substrate, (the key) to fit into it. This forms an enzyme-substrate complex and produces a product.
The diagram below shows how the enzyme and substrate work together to form a product:
I feel my results table and graphs clearly support my prediction. The average rate of reaction for urease concentration 0.3° is 0.0013300. This is a very slow reaction rate – it took around 12 minutes to complete the reaction, and this is evident because there is 5cm³ urea for only 1cm³ urease to act on.
Evaluation
I only encountered a small number of anomalous results in my investigation, but I came to the conclusion that these were down to human error and difficulty in being able to tell when the reaction has actually finished. This most distinct result which did not seem to fit the pattern was concentration 0.6° in experiment 2. Apart from this, the only other points which did not fit the pattern exactly were concentrations 0.83° and 1 from experiment 1. However, these results have not affects my overall outcome and simply show me that the method I have used is not perfect and can be improved upon which would, in turn, improve my results and conclusion. Despite this I still feel I have been able to draw a firm conclusion from my investigation.
I feel my results are accurate as I have repeated each experiment a total of three times, and almost all the results, apart from the aforementioned anomalies, are around the same figure. To increase the accuracy and to make my results and final conclusion more reliable, I have taken the average result for each concentration from all three experiments. This has ensured that the results I have collected are as accurate as possible and allows me to see the common trend forming. I think my experiment has been successful because the results I have collected clearly support my prediction, and as they have turned out the same each time, are reliable.
A downfall of the method is the fact that there is no actual way of telling the reaction has finished, so you have to guess when the solution is around the colour the experiment was stopped at for the last experiment. This is what I think has led to some inaccuracy within my results, because I had to estimate when the solution was the right pH. This experiment could be improved in a number of ways. I feel I have repeated my results enough times to achieve accurate results, but by using more concentrations of urease I would have produced a more accurate graph which would support my results further. I could have used concentrations higher than the concentration I started with, so that I could see how the rate changes through a greater range and to see if the maximum possible rate of reaction could be reached.