Procedure
The student will cut 2 cm by 2cm pieces of black and white filmstrip and place them specimen tubes for each temperature. The student will then measure out 20ml of 2% pepsin solution. I will add this to a specimen tube and repeat until all test tubes are full. I will put one test tube in a beaker containing room temperature water (testing the temperature at 22°c), one in a water bath heated to 42°c, one in a water bath heated to 54°c, one in a water bath heated to 61°c, and one in a water bath hearted at 79°c. To make sure the temperatures are constant, the student will place a thermometer in each tube of pepsin. The student will time each tube, checking every two minutes to see if the gelatin is digested by looking to see if the film is transparent. Every two minutes, all tubes will be shaken for three seconds. The film will only be counted as fully digested when it becomes completely transparent. I will repeat the experiment for each temperature four times.
To make sure the results are accurate, the student will:
- Obtain the pepsin solution with a measuring cylinder, the most accurate measurer available.
- Keep checking the temperature with a thermometer to make sure there are no large fluctuations.
- Have the thermometer in the pepsin and not the water so it records pepsin temperature.
Results
The results were put into tables rather than a graph due to the number of graphs needed for each individual temperature. If they were all plotted on one graph then the high average time for 22°c and the low average time for 79°C would make the graph seem out of proportion.
How the evidence was colleted with accuracy
This is how I made sure the experiment was completed with as much precision as possible.
- When the temperature was measured, the thermometer was placed in the pepsin and not the water surrounding it to get the accurate temperature of the pepsin.
- The thermometer was kept in the pepsin when the reading was being taken and was not taken out before the reading was taken.
- Pepsin levels were kept equal but were enough to make sure the film is totally submerged.
- The shaking of the test tubes was controlled – All four repeats were kept in the same rack and shaken for three seconds every two minutes.
- When cutting out the 2cm by 2cm filmstrip, lines were drawn on the filmstrip to ensure accuracy when cutting it out.
- The part of the filmstrip with holes in was removed and no piece of film with holes in was used, as the surface area would have been reduced.
- All four repeats were done at the same time to ensure they were all done at exactly the same temperature.
- A colorimeter was used to detect the ideal pepsin turbidity.
- The same person decided when the gelatin had fully dissolved so that the same perception was used.
Analysing The Results
The graph was plotted using the average times. A line of best fit was drawn on the graph so that we could see where the anomalous results were and which times other individual times would get if they were investigated. The graph clearly shows that temperatures increase the rate of digestion of Pepsin until a certain temperature where it denatures. We can see that the Pepsin denatures at around 68°C as after that temperature the time taken for the pepsin to break down the gelatin stays the same. By looking at the graph we can see that the Pepsin was very slow to work at 21°C and then, as the temperature increased, the time taken halved until it reached it’s optimum temperature. This is called the Q-10 factor and for every 10°C in rising temperature, the rate of reaction is doubled until denaturing occurs.
From this experiment I can conclude that temperature speeds up the rate of reaction until the temperature is too high and the enzyme denatures. This was as I expected because I know that temperature is a key factor in the activity of enzymes. If it is too cold the enzymes will move around to slowly, so the reaction rate is slowed. Likewise, if it is too warm they do not function properly. This is because the extra heat energy shakes them around so much that the active sites change shape so the enzyme molecules are denatured and can’t hold the substance.
These results partially support my prediction. The reaction time starts high, lowers until the optimum temperature, and then rises again after the enzyme denatures. The Q-10 factor has occurred, this is shown on the graph reading off the line of best fit, with 50°C giving a time of 16 minutes, and 60°C giving a time of 8 minutes. This shows that for every 10°C the rate of reaction doubles and the time to react halves.
The temperature increasing the rate of reaction can be explained using the kinetic theory. At a low temperature the atoms in the enzymes do not have much energy and are moving around slowly. As they are only moving slow they are not colliding with the substance as frequently and therefore they are not joining and reacting quickly. When the temperature is higher more energy is being supplied so they move faster and collisions occur more frequently between the substance and the enzyme.
As denaturing alters the shape of the enzyme, the lock and key mechanism cannot function correctly as they cannot bind together, thus rendering them useless.
Possible Errors In Data Collection
There could have been some errors whilst obtaining results:
- The film strip might now have been cut straight each time, meaning that there could have been more gelatin to digest on one than another.
- There might have been an error in the method with repeats. More repeats could have been done to give more accurate results.
- There might have been errors with checking the temperature. Thermometers might have been faulty.
- It is hard to keep the exact same temperature for the duration of the experiments
- There may have been a mistake when deciding if all the film had cleared.
- There will always be a degree of human error.
Anomalous Results
From the graph, there is one temperature recorded that does not fit in with the line of best fit. This is at 79°C. It is highlighted in a circle on the graph. This is could have been recorded wrongly because of any or all of the reasons listed in the “Possible errors in data collection” section. To correct this, I should have spent more time and I should have been more careful whilst doing the experiment. These results also question the integrity of the other results found as they were obtained via the same method.
Improvements To The Procedure
There are several things that I would improve if I repeated the experiment.
- I could give myself more time to do the experiment in so that I could carry it out in a calm environment and minimize the number of mistakes made.
- I could use a better timing method to obtain more accurate results, like taking a reading every minute instead of every two to gain more detailed results.
- Electric water baths used to improve their accuracy
- I could do a lot more repeats to improve the accuracy of averages.
- I could find better ways to measure and keep the temperature constant throughout the experiment so that mistakes would not be made with the temperature
Further Work
For further work, I would carry out and investigation to find the exact temperature for when Pepsin is denatured. I would find the point at which gelatin digested for each individual degree. This would help build up a near perfect solubility graph for the rate of which gelatin digested.
This experiment would be done with different percentages of Pepsin, ranging from 1% to 5%. This would show the effects of the different concentrations on the rate of digestion and the point of denaturing.
Gelatin could also be replaced by another protein such as egg white, agar jelly or regular jelly. Pepsin would still act as a protease. It would show how the difference in the structure of these proteins effects how the Pepsin acts in them and at what speed.
Trypsin and other chemicals could be used instead of the Pepsin to submerge the protein in. In the preliminary experiment, along with using Pepsin, we tried using water to submerge the gelatin filmstrip in but it had no effect, as water is not a protease.