Investigating The Effect of Temperature on Enzyme Activity.

The main factors that have to be fair, to allow a fair test to happen are the following:

• The same pH level- enzymes do not work well in extreme pH conditions.
• Enzymes are made from proteins- it is important that we do not get other chemicals on the film or in the trypsin and buffer solution.
• Lock and key- this is the means in which enzyme work. Shown below.

Substrate                                                         Broken down substrate

Enzyme  

• Denaturing of enzymes at different temperature- this will play a big part in the experiment, as it will determine how much of the substrate is broken down at different temperatures.
• Volume of solution
•  Concentration of buffer and trypsin.

In this experiment, I will be using the following temperature: 0°c, 20°c, 40°c, 60°c, and 80°c. The volume of the buffer and trypsin solution that I will use will range from 5cm³ to 10cm³ in multiple’s of five, the reason I have chosen these values is that they are round, easy to work with, and are easy to plot a graph with.  

Hypothesis:        

I believe that the rate at which the reaction will take place will slowly decrease after the temperature passes 40°c. this is because at 37°c, body temperature, enzymes work the best. Their reaction time is very low and so as 40°c is very close to this mark, the reactions will be at a peak. Before and after this, the reaction will take a longer time because the enzymes are denatured, and thus the lock and key cannot work.

Control Variable:        

In this experiment, water is going to be the control. This is because water’s pH level is 7, which is neutral. We should expect that no reactions should take place under these conditions, unless the water is biased. By this we mean that it has impurities, or enzymes present. For these reasons alone, we will be using distilled water.

Procedure for prelimary work

Aim:

To help us understand what is needed to complete the final experiment thoroughly, and so that we can understand exactly what is to be done.

Diagram:

 

Method:

• A temperature is to be randomly selected. For this work, I have selected 80°c.
• Take one test tube and put in 5cm³ of trypsin and buffer solution.  
• Place this in a water bath at a preheated temperature of 80°c.  
• Leave in the water bath for exactly 5 minutes so that it can acclimatise, and use a thermometer to measure the temperature of the solution and water.
• Once 5 minutes has passed, put in a split ended splint with a small piece of photographic film into the test tube.
• Leave the test tube in the water bath, and after every 30 seconds, check to see if the film has gone transparent.
• Have a cut off point of 10 minutes.
• Do these processes for 20°c, 40°c and 60°c, and take a record of each test.

Results:

Conclusion:

To conclude my prelimary work, I found out the following.

1. The acclimatisation period was just right, as it heated the test tubes to the right temperature, and would allow us to have four experiments for each temperature in the time provided. Also it is a round number, and easy to plot on a graph.
2. We found out that the volume of the solution being used was also correct, as in theory; the solution was made of equal parts of buffer and trypsin. Too much, or too little solution would drastically change the results we would expect.
3. The temperatures used are good because they are round, and in ratio to each other. This means that they are easier to plot on a graph.

Main Experiment

Aim:

To complete three tests and one control test of each temperature for a solution of trypsin and buffer.

Apparatus:

• 20 clean and dry test tubes
• 30 cm³ of water
• 80 cm³ of buffer and trypsin solution
• 20 split ended splints
• 20 pieces of equal sized film
• 1 stop watch
• 3 water baths set at pre-heated temperatures
• Results table

 Diagram:

Method:

1. Fill 15 test tubes of buffer and trypsin solution. The volume must be exactly 5 cm³ in each.
2. Fill 5 test tubes of water to act as a control for each experiment. The volume of these must also be 5 cm³.
3. Get 20 split-ended splints and fix a small piece of film onto the end of each one. Set aside.
4. Pre-heat one water bath to 80°c, one to 60°c and one to 40°c. leave some in a freezer, to be used in the 0°c test, and set room temperature at 20°c.
5. Put a thermometer into each test to see when the temperatures are right.
6. Take a temperature at random, for example 80°c. Put three test tubes of buffer and trypsin solution, and one test tube of water into the water bath, and let it acclimatise for 5 minutes. Use a stopwatch to make all the tests fair.
7. After the five-minute acclimatisation period is over, put a split-ended splint with a piece of film attached to it into each test tube.
8. Every 30 seconds, check to see whether the film goes transparent.
9. Record results accurately in a table.
10. Have a cut off point of 10 minutes.
11. Do stages 5-10 for each of the following temperature: 60°c and 40°c.
12. For 20°c, leave the four test tubes in the open in a test tube rack, and then follow stages 8 onwards.
13. For 0°c, put the four test tubes in a beaker full of ice, and then follow stages 8 onwards.

 

Results:

Graph:

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Analysis:

The results obtained via the experiments conclusively show that the temperature in which an enzyme works determines the rate of reaction. As you can see from the results table, the rate of reaction varies from 1.667 to 6.667. As there is no unit for rate of reaction, we must assume that this means that 6.667 of the substrate was broken down in so much time.

The graph shows, that in this particular case, a bell shape is produced. This means that from the temperatures tested, a gradually rise in temperature, from 0°c to 40°c increased the rate of reaction, but a further increase in temperature, from 40°c to 80°c decreased the rate of reaction.

From the graph, and the results table, we can clearly see that the optimum temperature, in this experiment, where the enzyme trypsin can break down a piece of film, is 40°c. This ties in with the scientific knowledge that the best temperature for the highest rate of reaction is body temperature, which is about 37°c. After this temperature, the enzyme slowly becomes denatured. This is backed up by the rate of reactions shown in the results table. They are:

At 0°c, the enzyme cannot work properly as there is not enough energy for the substrate to move around. The same happens at 20°c, but here, there is more energy present, allowing the molecules to move around slightly faster, but also so that not all the enzyme becomes denatured. At 40°c, the energy present is just right, and the molecules move around at high speeds, but also the enzyme does not become denatured. After this, the molecules move around too fast for the lock and key method to work, and the enzyme becomes denatured.

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Evaluation:

In this coursework, I have learnt many things upon how to complete a fair test, how to complete a safe and flaw proof test, and most of all, I have gained the scientific knowledge on how tyrpsin, and other enzymes work in different temperatures.

I believe that I carried out a safe test throughout the whole of the coursework. I used glass tubes, beakers, and thermometers in a safe and sensible way.

I think that all my tests were fair. This is because I used sterile, dry test tubes for all the tests, I used the same size piece of film for each test, an accurate thermometer and stopwatch was used, and new splints were used. A sterile syringe was used to improve accuracy. Also, the trypsin and buffer solution were of equal parts, and were not mixed with any other substances.

I feel that I recorded my results in an appropriate manner, using the right titles, space, and alignment. In the overall experiment, the right values were used, and also the right quantities of each substance.

To further emphasis the need for accuracy, I repeated each test three times to create an average, which would be more accurate than just a single test. This is because if an anomaly is found, then it would be easier to spot, and therefore easier to correct.

The main strength in this coursework of me was accuracy. I feel that I was always accurate, fair, and decisive. All the volumes, times, and calculations were done in a thorough and precise manner.

The only weakness that I had in this coursework was the fact that I wasted too much time in preparation, and also in between test. In the overall outcome, this accounted to about ten or fifteen minute, which are valuable in this type of circumstances.  

Improvements for other work include:

• Smaller time gaps in which to record a change in the state of the film.
• Less time to be wasted during and around tests. For example, acclimatisation periods.