Enzymes aid digestion; they convert large food molecules into smaller molecules inside your gut.
Prediction
I predict that as the temperature of the trypsin increases, the time taken for the substrate (film) to dissolve will decrease. However, as the trypsin reaches a certain temperature, I believe the film will take longer to dissolve. This is because as the protease reaches a certain temperature, the active site changes shape, or de-natures, meaning the substrate is not broken down at high speed. The active site no longer fits the substrate as shown below:-
As the temperature of the trypsin increases, the particles inside it will begin to vibrate a lot faster and gain more energy, enabling them to collide with other atoms a lot quicker. They also collide more frequently, meaning they smash against the gelatine’s atoms with more force, causing these particles to be broken down and dissolved a lot quicker. The particles become much lighter, meaning they can travel around a lot faster. The gelatine is contained on the photographic film and because gelatine is a protein, it will be dissolved by the trypsin, which is a protease.
Enzymes are affected a great deal by temperature. This graph shows the effect of increased temperature on an enzyme-controlled reaction.
The diagram on the next page shows that when the temperature of the trypsin reaches 40˚c, the protease’s active site breaks down and changes. Therefore the substrate no longer fits. We say that the enzyme has been de-natured and it no longer works. As the temperature jumps by 10˚c, the enzyme’s energy doubles. For example, when the temperature is at 20˚c, the trypsin has double the energy as it did at 10˚c.
Apparatus
Photographic film
Syringe
Trypsin
Water Bath
Thermometer
Stop Clock
Method
To ensure our experiment was fair, we only changed one thing, called the input variable. During our experiment, we changed the temperature but controlled a number of other things. These included the size of the photographic film, the water bath, the stop clock and the amount of trypsin we used. We cut the photographic film to 0.5cm² every time.
Firstly, we measured 1ml of trypsin using our syringe and added it to the test tube. We heated the trypsin to the correct temperature and measured accurately using the thermometer. We made sure that enzyme was at the required temperature before we added the film. When the enzyme was at the correct temperature, we added the photographic film to the test tube, covering it fully with the trypsin. As soon as the photographic film was underneath the trypsin, we started the stop clock. We checked to see whether the film had dissolved regularly, roughly every 30 seconds. When the film had fully dissolved, we stopped the clock. We carried out the same procedure with each of our readings, using the same amount of trypsin and photographic film.
To ensure accuracy, we took repeat readings for each of our temperatures and calculated an average. We will also read the thermometer at eye level ensuring we gain a accurate temperature.
Results
Conclusion
During our experiment, we found out a lot about the way trypsin behaves under different conditions. We learnt that as the temperature of the trypsin increased, the time taken for the photographic film decreased. I predicted this, although I also thought that when the trypsin reached a certain temperature, the active site would de-nature, causing the time taken for the film to dissolve to increase. Whilst carrying out our experiment, we did not find this. We found that as the temperature increased, the time taken for the film to dissolve decreased, but it did not reach a point when the time taken increased. Instead, for the whole of our experiment the time taken decreased as the temperature increased, giving a negative correlation.
There are many reasons as to why this might have been the case. Firstly, the trypsin may not have reached a great enough temperature for the active site to de-nature. This would have meant that the substrate would have kept fitting the enzyme, but due to the increase in the molecule’s energy, the substrate would have been dissolved at a very quick rate.
We found this pattern due to the behaviour of proteases: The enzymes worked on substances called substrates. The reaction took place on a part of the surface of the enzyme called the active site. The active site is where the substrate enters so that the products can be released. The active site must be a certain shape to fit the substrate trying to enter. For example, if the substrate has straight, smooth sides and so does the enzyme, then the substrate will be allowed to be broken down by the enzyme. However, if the substrate is very rough and jagged, when the active site is smooth and straight, the substrate will take a lot longer to get broken down. When enzymes reach a high temperature, their active site de-natures and they are unable to break down molecules as quickly as they were.
As the temperature of the trypsin increased, the particles inside it will began to vibrate a lot faster and gained more energy, enabling them to collide with other atoms a lot quicker. They also collided more frequently, meaning they smashed against the gelatine’s atoms with more force, causing these particles to be broken down and dissolved a lot quicker.
The error bars that I have drawn on my graph show the range and accuracy of the readings that I took. The longer these bars are, the less accurate my readings were. My first and third readings looks very inaccurate compared to the rest of the readings. This could have been because we were not paying enough attention and focusing on other things while taking these readings. It also may have been because different people were recording the time taken for the film to dissolve, meaning different people’s thoughts as to when the film had fully dissolved varied.
I have drawn a dotted line on my graph to represent where the film should have started to take longer to dissolve due to the active site becoming de-natured under the intense heat. However, this was not the case in our investigation. As the temperature of the trypsin reached 40˚c, the photographic film should have taken more time to dissolve than at 37˚c. This is because after the protease has reached a certain temperature, it stops working properly due to the change in shape of the active site. Although the time taken for the film to dissolve does increase here, the film does not stop dissolving. Instead, it just takes a longer time.
When I compared my results with other people, I found that my results were fairly accurate. However, many of their results seemed to be starting to increase in time taken for the film to dissolve at roughly 40˚c.
Evaluation
I was generally very pleased with our method and the way in which we carried out our experiment. However, as we were collecting together our results and timing how long the film took to dissolve, a number of things were brought to our attention that could have affected the fairness of our experiment. Human error had a big part to play in the inaccuracy of our experiment.
Firstly, it was very difficult to see when all of the film had been dissolved. Many people may have thought it had dissolved at different points, so stooped the clock at different times, giving inaccurate results. We could have used a light sensor to measure accurately as to when all of the photographic film had been dissolved. As more of the film and gelatine had dissolved, more light would have been able to pass through the film. As this happened, the light sensor would have alerted us as to when all of the film had been dissolved. Secondly, it was also difficult to measure the exact temperature of the trypsin although we did use a thermometer. The temperature of the trypsin also decreased after we took it out of the water bath. This meant that the trypsin was not at the same temperature throughout the experiment, meaning there were more than one input variable in our experiment.
I believe our results were fairly accurate compared to other people’s. However, our last three readings were anomalous. We recorded three readings for every temperature to ensure accuracy, yet this error bar was particularly long in comparison to the rest.
If I were to carry out the same experiment again, there would be a number of things I would change and do differently. I would use more temperatures, so that hopefully the graph would begin to go upwards. , we could take more than three readings to make our results even more accurate. Although three readings are enough to produce a graph, we might be able to draw a better conclusion. As people moved around and breathed around the test tubes as the film was being dissolved, excess heat may have been passed on to the test tube or into the trypsin. This could have caused the temperature of the trypsin to increase, giving inaccurate results. We could use a troff filled with cold water and put it into a beaker with the photographic film inside the test tube. This would hopefully absorb all of the heat being circulated in the air and stop it from tampering with our results.
