-Timer x5
-Test tube x5
-Test tube rack
-Scissors
-Ruler
-Stirrer
-Photographic film
-Beaker of 5% Trypsin
-Syringe x6
-Buffer solutions in the following pHs: 5, 7, 8, 9, 11
Method:
-Firstly I will cut the photographic film up into 15 equally sized pieces (1cm squared). Put one piece into each of the 5 test tubes.
-Secondly, I will measure out 3cm cubed of a buffer solution into one of the test tubes. I will do this for each of the five buffer solutions, making sure to use a clean syringe for each one so as not to contaminate them. I will make careful note of the buffer solution I have put into each test tube by labelling the tubes with their pH.
-I will then use a clean syringe to measure out 1cm cubed of Trypsin into each of the test tubes, starting the timers simultaneously.
-Every minute, I will stir each test tube 3 times so it doesn’t affect my results.
-I will check each tube continuously to see exactly when the film goes completely clear and colourless.
-When the film has gone completely colourless, I will stop the timer instantly.
-I will repeat the experiment three times to get more accurate results.
-I will record my results into a table clearly.
Safety:
-I will use a lab coat to protect my clothing.
-I will use goggles to protect my eyes.
-I will use gloves to protect my hands.
Fair Test:
I will keep this a fair test by:
-Wearing gloves so as not to contaminate the experiment with any substances on my hands.
-I will use different equipment for each pH so they don’t get contaminated from one another.
-I will stir the test tubes in a consistent manner so that it doesn’t affect my results.
-I will perform each of the tests at the same temperature to the best of my ability.
Scientific Background Research on Enzymes:
An enzyme is a biological catalyst. This means that it is an organic substance made by the body, which speeds up the chemical reaction to break down food that takes place in the human body. By definition, a catalyst does not get used up in the reaction. Most enzymes are proteins.
All enzymes have an active site. This is where the Lock and Key theory can be explained. The active site is an area on the enzyme molecule where the reaction takes place. The active site of each enzyme is unique and only the substrate, which the enzyme is designed to break down, will fit into it. The enzyme’s active site and the molecule of the substrate fit together like a key in a lock.
The more concentrated the enzyme, the faster the reaction. This is to do with kinetic theory as the greater the number of enzyme molecules around the substrate, the more likely that a high-energy collision will occur, meaning that the reaction will be faster.
The temperature of the surroundings has an effect on the enzyme’s activity. The higher the temperature, the faster the reaction is. This is true up to a certain point. Again kinetic theory is used to explain this: when the enzyme molecules are hot they move around more and a lot faster, this means that a collision with the substrate is much more likely. This is only true up to a certain temperature (very high or very low), when the enzyme becomes denatured. This means that it can no longer work because the active site’s shape has changed.
Lastly, all enzymes have an optimum point. This is a set of conditions that it works best at. For the enzyme that will be used, Trypsin, this is pH 8 and roughly 37.5 degrees Centigrade.
Prediction:
I predict that the photographic film will go clear in the quickest time when the test is performed at pH 8, because in my research I found that Trypsin likes to work at this pH.
I also predict that as the pH gets further away from 8, in both directions, the time it takes to react with the gelatin will increase.
I think that Trypsin will only react with the gelatin and nothing else as, according to the Lock And Key theory.
I believe that my results graph will look like this:
Obtaining Evidence Section
Table:
An example of how the average time was calculated:
pH 7
689 + 696 + 686 = 2071
2071 = 690
3
An example of how rate was calculated:
Rate = 1
time
pH 7
Rate = 1 ÷ 690
= 0.001449275362
= 0.00145 (to 5 decimal places)
Analysing Evidence Section
Patterns and trends:
My graph of rate shows that as the pH increases, or as the alkalinity increases, so does the rate of Trypsin’s activity. This means that it is a proportional relationship. The rate increases by 0.00257 s between pH 5 and pH 11. The average difference in rate between each pH is 0.0006425 s ,however it increases by a different amount each time.
From my results I conclude that Trypsin works the quickest under alkaline conditions.
My prediction:
My prediction was inaccurate. I predicted that Trypsin would work the fastest at pH 8 and become denatured at any extreme pH. This did not happen; Trypsin continued to work at the pH’s between and including 5 and 11, which are very acidic and very alkaline respectively. However, my results do support my prediction slightly in that the rate of Trypsin's activity increased at pH 8. Altogether, my results don’t support my prediction very well.
Conclusion:
I conclude that the rate of Trypsin's activity increases as the alkalinity increases. I also conclude that the point at which Trypsin becomes denatured must be either above pH 11 or below pH 5 or both. When Trypsin becomes denatured, it will stop breaking down the gelatin, however it did in all of the pHs I tested, so it was not denatured.
Evaluating Evidence Section
Improvements To The Method:
I would recommend my method to others to use as it was simple and provided me with enough results to plot a graph with. I think that the method was not without fault, however as I found some things inefficient.
For example:
- I would use smaller pieces of photographic film. This is so that I wouldn’t have to wait as long for the Trypsin to break it down.
- I would have used more liquid of either buffer solution or some added distilled water. This is because, as the enzyme breaks down the gelatin on the film, the black colour comes off. It then clouds up the solution and makes it difficult to see the point at which the film turns clear. With more solution, the colour would be less concentrated. If it affected the result it would not have been a problem, as the same would be added to every test.
- I would not perform each of the tests simultaneously, as it is difficult and often inaccurate to be watching all the tests at once.
- I would do at least 5 repeats altogether. This would make the margin for error much narrower. If I do the tests 5 times then take an average it will be much more accurate.
Limitations:
I split the practical over two days. As I was performing it at room temperature, this is a limitation. I did not take the temperature on either day. This means that it could well have been colder on one day than the other. As temperature is a factor affecting Trypsin’s activity, it is not a fair test, as it was not kept constant.
In the actual laboratory that I was doing the tests in, the temperature might have varied as there are parts of the room that are colder than the rest e.g. near the window and draughts.
The photographic film was not measured and cut with absolute precision so it may have been different sizes.
The Accuracy Of My Results:
I think that, given the materials I had available to work with, m results were fairly accurate. There were no anomalies in my results. The fact that I did some repeats meant that there was a greater chance of my results being correct.
The Extension Of This Investigation:
To extend this investigation I could further prove my conclusion by testing the rate of Trypsin in the same pHs but also investigate the ones in between the whole numbers. Eg look at the rate of the following pHs: 9.0, 9.2, 9.4, 9.6, 9.8, and 10.0. This way it would show my theory in greater detail.