Rate of an enzyme catalysed reaction plotted against substrate concentration [S]
Source: http://www.schoolscience.co.uk/content/5/chemistry/proteins/Protch6pg2.html
Safety in the lab
- Wear safety goggles because it could be dangerous if any substances were to make contact with your eyes.
- Have something ready to wipe up any spillages.
- Make sure any cuts are covered up with plasters because it could be dangerous to have any substances enter your body through a cut.
- Make sure there is some eyewash ready in case something gets in your eyes.
- Handle heating equipment with care after use to avoid burns.
- Pick up heated test tubes with a test tube holder, also to avoid burns.
Fair testing
- Keep the concentration of the trypsin constant throughout the whole experiment.
- Keep the concentration of the milk constant throughout the whole experiment.
- Use the same volume of milk for every test during the experiment.
- Use the same volume of trypsin for every test during the whole experiment.
Plan Experiment
In order to accurately investigate how temperature affects rate of reaction, I must conduct a plan investigation to find out the appropriate volumes and conditions to be used in the main experiment.
I will use powdered milk, which contains a white protein called casein. When trypsin is added to the milk solution (powdered milk and distilled water), the casein in the milk will be broken down and therefore the solution will turn clear.
Method
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I will add 2cm3 trypsin to a powdered milk solution in a test tube (2cm3 at room temperature, and also to powdered milk in a test tube in a water bath.
- Start the stop clock.
- Watch the solution, when it turns clear, stop the clock and record the time.
Improvements and observations
After conducting the trial experiment, I noticed that I needed to control the temperature and record it, not just using vague values such as room temperature and hot water in a water bath. Also, I only took two results, which isn’t enough to make any good conclusion from, and therefore I will take more readings. In the main experiment, I will conduct the experiment at the following temperatures (°C): 15, 25, 35, 45, 55, 65, and 80. I will only go as far as 80°C because at this temperature I expect that the temperature will have denatured all of the enzymes. A denatured enzyme will not work, and the process cannot be reversed.
For the main experiment I will take 3 readings instead of 1, so I get can more reliable results, and take an average. This means that if there is an anomalous result it is obvious, and I can still get an average from the other 2.
I also will control the other two factors affecting enzyme rate of reaction: concentration and pH level, so I can focus on temperature. Using the same batch of powdered milk throughout the experiment will control PH. Concentration of both substrate and enzyme will also be controlled because I will use the same volume and batch of each. To achieve this as accurately as possible, I will use syringes to measure the quantities of each, keeping a separate syringe for the milk and one for the trypsin, this is to avoid potential contamination.
Another problem experienced in the trial experiment was deciding what counted as “clear”, as this is ambiguous. This was because although the solution was supposed to go clear, it never quite became absolutely colourless, thus I will have to control this by stopping the experiment once it gets to a certain shade.
Hypothesis
Enzymes work faster when it is warmer due to kinetic theory, that is, the particles have more energy, therefore they move faster so it is more likely a substrate will come into contact with an enzyme hence there will be more reactions per second. The accepted relationship between temperature and rate of reaction is that for every 100C temperature rise; the rate of reaction will double. Yet this doesn’t occur indefinitely, after approximately 38oC, the active site changes shape so the substrate no longer fits, thus the enzyme is denatured. Therefore, I predict that the enzymes will work best at a temperature in the region of 35 to 400C. I also predict that up until the optimum point, the increase in the rate of reaction will conform to the “Q10 = 2” theory, which means that the rate of reaction will double with every increase of 10o.
Results
Analysis and Evaluation
From these results we can see there is a clear anomalous reading in the first result at 55°C. There are also two less clear anomalies for the last reading at 15°C and the final reading at 65°C. Here is a table with the anomalous readings omitted:
The omitted results are shaded.
The graph on the following page shows both sets of results.
Omitting the anomalies made a difference to the final results, and the optimum temperature changed because of the omissions, from 65o to 55o. This shows that it is important to take out anomalous results.
The “Q10=2” theory fits fairly well. At 20°C, the rate is approximately 0.0035 and at 30°C, the rate is 0.0067, which is almost double. Between 25°C and 35°C, the rate approximately doubles from 0.004918 to 0.008696, which backs up this theory.
However, there is a big difference between the hypothesis and the results. This is the peak temperature for enzyme reactions, which was predicted at roughly 35°C to 40°C, was in fact about 55°C, a very different result. The enzyme trypsin comes from a fungus, not the human body; therefore its peak temperature is not roughly 38°C, which is body temperature, and so I don’t think this difference is down to poor experimenting, but more down to poor judgement.
Although that part of the hypothesis was incorrect, the rest of the hypothesis concurs with the results. After the peak temperature, the enzyme clearly denatures as the rate quickly drops, and at 80o there is no result, which proves this. However, I didn’t take any results between 65 and 80o so it may have completely denatured earlier.
Generally, I believe that the results collected were reliable. This is because it was planned, and improved, thanks to the trial experiment, and then carried own in a controlled way, with repeats. Despite the anomalies, of which there were little, I think that a reasonable analysis could be made, and there were enough results to draw a fair conclusion from. However, there were some areas where more results would have been beneficial. Between 65o and 80o I should have taken at least one more results to determine where the enzyme finally denatures. Also, lower temperatures would have been useful to establish at how low temperatures trypsin works. Finally, more results around 55o would have been helpful to ascertain the peak temperature more accurately, as currently I can only assume that trypsin works best at 55o.
From this experiment we can see that enzymes don’t all act in the same way, and that trypsin doesn’t act in the same way as some human enzymes. However, from the shape of the graph, that the change in rates of reaction is similar between enzymes, and that the “Q10 = 2” theory applies. Also, although I don’t have enough results to confidently state the peak temperature, but I can say that it is approximately 55o and also that trypsin is denatured by 80o.
To further the investigation into trypsin, I could investigate another factor that affects it, such as pH or concentration. I could use the same method, but use less or more of the substrate or the enzyme to see how concentration affects the rate of reaction. I could also keep concentration and temperature the same and affect the pH of the casein, which would determine the peak pH for the enzyme trypsin.
