The equipment that shall be used will be test tubes, beakers, stopwatch, and a thermometer. The times will be recorded 3 times and then an average taken for the result. The Trypsin and milk protein will be put in two separate test tubes and then after 5 minutes, they will be mixed. The trypsin and milk protein need to be at the same temperature, otherwise it could effect the results. All three tubes will be mixed and put into the water at the same time. This will ensure that the results will all be fair. In addition, it is vital that the trypsin is always added to the milk and not the other way round, if it happens the other way round then the results could be biased. To know when all of the protein has been digested there needs to be a control, which has previously, be digested. This is to compare against and determine when all of the protein has been digested.
The Key Factors in this investigation are that throughout the experiments, the temperature stays constant and that the amount of milk protein and trypsin is always the same. The temperature will be changed for each case and the time taken for the protein to be fully dissolved will be measured.
The process will be repeated three times and then the average will be used for the results. This will ensure a fair and more accurate result.
I have decided to take a measurement every 10 oC from 10 oC to 60 oC. In addition, a measurement at 100 oC will take place to show a definite denatured result.
In preliminary experiments I found that the protein and trypsin were at different temperatures to start with – I decided to put the test tubes into the test water for 5 minutes before the experiment; this is to get them both at the correct temperature. As the experiment went on, due to it taking a relatively long time, the temperature in the beaker changed temperature – I decided to monitor the temperature and add hot water/ice to keep the temperature constant.
Here are the results that I got: -
M1, M2, M3 = Measurement 1,2 & 3
The results made this graph: -
The rate of reaction was found by
1/average x 10-3
Eg. 1/659 = 0.0015174
0.0015174 x 10-3 = 1.5174
My results show that temperature does effect the rate in which trypsin breaks down protein.
From the graph, I am able to back up my theory that as the temperature increase, so shall the rate of reaction. I can see when the enzyme is most active and when it starts to denature. From the graph, I have found out that, as the temperature increases, so does the rate of reaction, as the enzyme and substrate have more kinetic energy and, up to a certain point (~60°C) where the activity ceases altogether. I found that the optimum for trypsin is at 40°C. This is where the greatest number of collisions takes place between the enzyme and the substrate and therefore the highest rate of reaction is.
The rate was higher at the higher temperatures (up to 40°C) because as the temperature is raised, so is the energy level of the enzymes and substrate molecules. This means that they have more kinetic energy so they collide more often and therefore more reactions take place between them. This, in turn, means that the rate increases as more polypeptides are produced.
The enzyme denatured at about 60°C because the weak bonds, which hold the molecule into the specific shape for one substrate, are broken. The increase in collisions and vibrations at high temperatures is great enough to permanently change the shape of the active site. The enzyme is said to be denatured because it can no longer break down specific molecules because its active site is permanently disfigured.
My prediction was correct in that there was very little activity at 10oC because the speed at which the enzymes and substrate molecules were moving was very slow so there were not many collisions between them. The optimum temperature was not the same as I predicted at 40°C instead of 50°C. The temperature at which the enzyme denatured and the activity stopped, was quite far off my prediction because the optimum temperature was lower than I predicted nearly the same temperature at 40/50°C instead of 60°c as was predicted.
The evidence did not turn out exactly as I expected. Most of the temperatures that I found were lower than I predicted; this could have been because of a number of reasons. The thermometer used was the same for all of the tests, that thermometer could have been slightly wrong. Also, the temperature of the water bath it was in could have changed without me knowing.
I believe that my method gave evidence that is quite reliable in the angle that it shows that temperature does effect Trypsin and its break down of proteins. But, the actual values are not reliable, I believe that an error occurred in the investigation which made the enzyme denature at a lower temperature.
I believe that although the rates were not quite correct for the temperatures, all of the results were similar and fitted the main stream of results and there were not any anomalies. I think that the way in which I determined when the trypsin had broken down all of the protein was not very accurate. By comparing, it to a control may mean that the protein may not have been fully digested when the time was noted down. At the low temperatures, just the slightest f differences in appearances could take 30 seconds that would make a considerable difference to the results. Although the method for determining a fully broken down test-tube of protein wasn’t very accurate I cannot see any other way of doing it with the limitations of a secondary school laboratory.
There was enough evidence to draw a sound conclusion and extending the investigation further would make the results more accurate.
If the investigation were to be repeated I would use a digital thermometer linked up to a computer so that the temperature can be closely and accurately monitored, this would give results that are a lot more accurate. Although the method for determining a broken down protein was quite in accurate, it is the only way. But, I would note down when I think it has finished, keep the stopwatch timing and if any further change is seen, to note that time down and to continue with that method until it is totally finished.
Maybe a different protein could be used and see if better results are found which would make a better conclusion.
On the whole I feel that the investigation was a success in showing that temperature does effect Trypsin and it breaking down protein, although the numerical evidence was not quite sound.
