Investigation

Investigate the effect of temperature on the rate of a reaction catalyzed by the enzyme trypsin.

Plan

Hypothesis

To investigate the effect of temperature on the rate of reaction of the enzyme trypsin and from this, determine the enzymes optimum temperature.

Introduction

Enzymes are globular protein molecules, which are often defined as biological catalysts. They catalyse the breakdown of a substrate into two or more product molecules, using the cleft in its surface called an active site. The substrate molecule has a complementary shape. The random movement of the enzyme and substrate brings the substrate into the active site. An enzyme-substrate complex is temporarily formed. (1) This is because the R groups of the amino acids in the active site interact with the substrate and this interaction breaks the substrates apart. The two product molecules then depart the active site, leaving the enzyme molecule unchanged and ready to bind with another substrate molecule.

Trypsin is a digestive enzyme, known as a protease, secreted by the pancreas. Trypsin attacks peptide bonds of proteins and polypeptides, thereby converting large peptide chains into shorter polypeptide segments. There are two basic types of proteases, the endopeptidases and the exopeptidases. Trypsin is an endopeptidase that is found in the alimentary canal of nearly all invertebrates and vertebrates organisms that digest in this way. It is specific for digestion of peptides and esters of the amino acids lysine and arginine. The enzyme attacks bonds located deep within the substrate protein, thus transforming large peptide chains into shorter polypeptide segments.

Powdered milk (Marvel milk) contains a white phosphoprotein called casein. It is found in milk as a suspension of particles called micellae, which are held together by calcium ions. When the enzyme, trypsin, is added to its substrate, the amino acid chains that are located in the protein; casein, will be broken down and white opaque suspension becomes translucent.

Prediction

I predict that at low temperatures the reaction will take place relatively slowly. At lower temperatures, the molecules will have less heat energy to be transferred into kinetic energy and as a result, the molecules will move more slowly and binding between the substrate and enzyme is less common. This is because the substrate molecules will not often collide with the active site of the protein. When molecules collide, the kinetic energy of the molecules can be converted into chemical potential energy of the molecules. The chemical potential energy of the molecules at low temperatures is relatively small because they do not often collide and so a change in chemical state will result much more slowly, thus the reaction will too, take place more gradually.

As the temperature rises, the enzyme and substrate molecules move faster. This is because collisions will happen more frequently which results in the substrate molecules binding with the active sites more often. When they do collide, they will do so with more energy. This makes it easier for bonds to be broken so the reaction can occur. Thus the greater the kinetic energy of the molecules in a system, the greater is the resulting chemical potential energy when two molecules collide. As the temperature of a system is increased it is possible that more molecules will reach the activation energy more rapidly and the rate of the reaction will increase. As the temperature increases further, the speed of the movement of the substrate and enzyme molecules also continues to increase until an optimum temperature is reached. The optimum temperature is the temperature at which enzymes catalyzes a reaction at the maximum rate.

Most human enzymes have an optimum temperature of around 40°C. By keeping our bodies at temperatures around 37°C, it is ensured the enzyme catalayzed reaction occurs closest to its optmum temperature. The trypsin used in this experiment is obtained from bovine (cow) pancreas. Since cows are mammals like humans, the enzyme is likely to have an optimum temperature similar to human trypsin, approximately 40°C.

Above around 40ºC I predict to see a reduction in the rate of reaction. As the temperature is increased, past this temperature, the internal energy of the molecules will increase. This internal energy may include the translational energy, vibration energy and rotational energy of the molecules and the energy involved in chemical bonding of the molecules as well as the energy involved in nonbonding interactions. (2) This will cause the molecule to vibrate vigorously. Some of this heat may be converted into chemical potential energy. If this chemical potential energy increase is great enough, some of the bonds between the chains of amino acids that determine the three dimensional shape of the active proteins will be broken due to this vigorous vibration. This could lead to thermal denaturation of the protein and thus inactivate the protein. The enzyme molecule will begin to lose its shape and initially the substrate molecule does not fit as well into the active site of the enzyme and the rate of the enzyme catalyzed reaction will decrease. Eventually, the substrate or enzyme will no longer fit at all, or can no longer be held in the correct position for the reaction to occur.

This can be visualised with the ‘lock and key theory.

There are a number of other factors that affect the rate of reaction.

Because I know these will affect it, I will keep them constant.

Method

To investigate the effect of temperature on the rate of reaction and discover the optimum temperature, Marvel milk suspension and trypsin solution will be used. Trypsin solution and Marvel Milk suspension will be heated, to the same specific temperature in a water bath. The two solutions will then be mixed together and timed for a period, before the mixture is placed into the colorimeter to measure the level of hydrolysis it has undergone. The procedure will be repeated at a range of temperatures.

Preliminary Work

Preliminary work will be used to investigate the amount of time the Marvel milk suspension and trypsin solution should be left to hydrolysis for. From my scientific prediction, I expect the optimum temperature to be around 40°C. Therefore I will conduct all preliminary work at the predicted optimum temperature as this will provide the fastest rate of hydrolysis that my experiment is likely to undergo.

From the experiment I can see that from 150 seconds and above, the rate of reaction is so fast, it appears that the colorimeter is not measuring a dramatic difference ...

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Preliminary Work

From the experiment I can see that from 150 seconds and above, the rate of reaction is so fast, it appears that the colorimeter is not measuring a dramatic difference in the absorbance. This is because the solution has undergone complete hydrolysis and the solution has cleared. Therefore this solution has been left for too long. Below 60 seconds the solution has not hydrolysed substantially enough. Bearing in mind this is expected to be the fastest level of hydrolysis, it appears this period would not be long enough when the temperature is reduced. Because of this reason I will leave the solution for 120 seconds. This appears long enough to measure a suitable amount of hydrolysis.

Apparatus

100cm3 Marvel milk suspension:

4g Marvel Milk to 100cm3 distilled water – this will be an adequate amount for this experiment.

100cm3 trypsin solution:

0.5g trypsin dissolved in 100cm3 distilled water – this will be an adequate amount for this experiment.

5cm3 0.1M hydrochloric acid – this will be an adequate amount for the experiment
Thirty eight flat-bottomed test tubes, each approximately 2 x 8cm – the test tubes are able to be heated to the desired temperatures and the size is adequate for 10cm3 of solution.
Three 10cm3 plastic syringes – this will make the measurements of the hydrochloric acid, marvel milk suspension and trypsin solution more precise
Two 150cm3 glass beakers – the marvel milk suspension and the trypsin solution will be held in these beakers
Stop clock – to time the reaction exactly to two minutes
Colorimeter – measure the level of hydrolysis precisely
Top hand balance – measure the quantity of the marvel milk and trypsin precisely to the nearest 0.5 grams
100cm3 measuring cylinder – measure out the quantity of distilled water precisely
Two spatulas – handle the milk powder and trypsin safely
Calculator – calculate the averages
Surgical gloves – for use with hydrochloric acid, as it is corrosive
Thermometer – precisely measure the temperature of the water baths
Water baths maintained at:

20°C
30°C
40°C
50°C
60°C
70°C

Risk Assesment and Precautions

Trypsin should be stored at very cold temperatures (between -20°C and -5°C) to prevent denaturation.
All quantities need to be measured out precisely using the required equiptment to ensure a fair test.
Some people are allergic to enzymes, so I will be careful to mop up any spillages and will wash my hands after the practical work.
I will take care when using the water baths as they will be reaching high temperatures, and will keep all books and belongings away from all apparatus.
The milk suspension is harmless and presents a low risk, but the 0.1 M hydrochloric acid is corrosive. When using the hydrochloric acid I will wear surgical gloves.
I will wear safety goggles throughout the experiment to stop any solution reaching or entering the eye.
I will tie my hair back for a clear and safe view of the process and to ensure it does not enter any solutions and contaminate them.
I will take great care throughout the experiment, making sure I do not put myself or others in any danger.

Main Method

Using a top hand balance and spatula, measure 4g Marvel milk powder into a glass beaker.
Using a measuring cylinder measure 100cm3 distilled water and add to the 4g Marvel milk powder.
Using a top hand balance and spatula, measure 0.5g trypsin into a glass beaker.
Using a measuring cylinder measure 100cm3 distilled water and add to the 0.5g trypsin.
Introduce 5cm3 Marvel milk suspension and 5cm3 distilled water into one flat-bottomed tube using a plastic syringe. This is the colour standard, which will be used to indicate the absence of any enzyme activity.
Use the colorimeter to measure the colour standard and record.
Introduce 5cm3 Marvel milk suspension and 5cm3 hydrochloric acid into one flat-bottomed tube using a plastic syringe. This is the colour standard to show complete hydrolysis.
Use the colorimeter to measure the colour standard and record.
Introduce 5cm3 Marvel milk suspension into each of the eighteen flat-bottomed tubes using a plastic syringe.
Introduce 5cm3 trypsin solution into each of the eighteen flat-bottomed tubes using a plastic syringe.
Transfer one tube containing Marvel milk suspension and one tube containing trypsin solution into a water bath at 20°C, checking its temperature with a thermometer.
Allow ten minutes for the contents of each tube to reach the temperature of the water bath.
Mix the contents of the test tube containing the trypsin solution to the Marvel milk suspension and start the stop clock.
Time the hydrolysis for 120 seconds and transfer solution immediately into the colorimeter and measure. Record this result into the table.
Repeat stages 11 to 14 using water baths at temperatures of 30°C, 40°C, 50°C, 60°C and 70°C.
Repeat stages 11 to 15 twice more, recording all results in the form of a table.
Using a calculator, calculate the averages of the results for each temperature and record into the table.

During the experiment there are a number of variables that need to be kept constant:

Agitation. This would aid the random movement of the substrate and enzymes within the solution and so will need to be kept constant. I have therefore decided to only stir the solution three times in a clockwise direction, when the enzyme is added to the substrate.
The concentration of milk suspension and trypsin solution need to remain constant, as varying this would dramatically alter the results. This is because Therefore an accurate syringe will be used to measure the exact amount.
The temperature needs to correspond to what is required. To do this, a thermometer will be used to check the temperature of the solutions before and during the experiment to ensure that it remains constant.
Pressure will affect the results of the experiment but the experiment will be carried out at room pressure, which will not be subject to any large pressure changes.

Analyzing Evidence

Results

Table to Show Colour Standards of Distilled Water and Marvel Milk Solution and Marvel Milk Solution and Hydrochloric Acid

Colour Standards are used to measure the levels of complete hydrolysis (Marvel Milk Solution and Hydrochloric Acid) and no hydrolysis taking place (Distilled Water and Marvel Milk solution). These results can then be compared to the levels of absorbance between the Marvel Milk and Trypsin Solutions, in the analysis and evaluation.

Table to Show Initial Results measuring the Absorbance Rate of the Marvel Milk and Trypsin Solution in comparison to the Temperature

Drawing Conclusions

Discussion

The distilled water and marvel milk solution is a colour standard, which can be used to determine the absorbance when hydrolysis is not taking place. From my results I can see an obvious trend between my prediction and outcome of the experiment. At 20°C, the measurement made in the colorimeter had an average absorbance of 0.50A. At this low temperature, the reaction is taking place relatively slowly. (The solution is still hydrolysing because of the trypsin, as the absorbance is 0.61 when there is no hydrolysis taking place) This is because the molecules have less heat energy to be transferred into kinetic energy and as a result, the molecules are moving more slowly. Therefore, the binding between the casein and the trypsin is less common. This is because the substrate molecule did not often collide with the active site of the protein. The chemical potential energy of the molecules at low temperatures is also relatively small also and so the enzyme catalysed the reaction much more slowly.

As the temperature increased by 10°C, to 30°C, there is an obvious increase in the rate of reaction, with an average absorbance of 0.12A. The trypsin and casein molecules move faster. This has resulted in more collisions happening more frequently, which has resulted in the substrate molecules binding with the active sites more often. When they did collide, they did so with more energy. This made it easier for bonds to be broken so the rate of the reaction increased. The greater the kinetic energy of the molecules in a solution, the greater the resulting chemical potential energy when trypsin and casein molecules collide. As the temperature increased, more molecules reached the activation energy more rapidly and the rate of the reaction increase.

At 40°C, the speed of the movement of the casein and trypsin molecules have continued to increase and more collisions have surcum as a result. In comparison to 50°C, it appears that trypsin is catalysing the reactiuon at its maximum rate therefore it can be concluded that the optimum temperature has been reached. This is when all the typsins active sites are being used up, as the collisions are happening so fast almost momentarily after an active site becomes empty, it is filled with another casein molecule. When compared to the hydrochloric acid and marvel milk solution, there is a difference of 0.03A. As this difference is very little, I am able to see my results are reliable. Although it does highlight that at 40°C the optimum temperature hasn’t been completely reached, so this could be the basis for an extension experiment.

Beyond 40°C the enzymes have begun to denature. This is seen in my results, when the average absorbance at 50°C was 0.10A; greater than the previous absorbance at 40°C of 0.07A. Above 40ºC, as predicted, there was a reduction in the rate of reaction. This is because the internal energy of the molecules has increased. This internal energy caused the molecules to vibrate vigorously. Some of this heat was converted into chemical potential energy and this chemical potential energy increase was great enough that some of the bonds between the chains of amino acids were broken. As a result the enzyme molecules begun to lose their shape and initially, the substrate molecule did not fit as well into the active site of the enzyme. This denaturing of the enzyme became more serious as the temperature increased, which can be seen in the graph.

At about 70°C, the substrate and enzyme could hardly fit at all, or could no longer be held in the correct position for the reaction to occur. There is also still a reaction taking place at these high temperatures, where I would expect the rate of reaction to have decreased more substantially than it has done. This is most probably because casein consists of a fairly high number of proline peptides, which do not interact which means that there is less scope for the molecules to be broken down. There are also no disulphide bridges. As a result, it has relatively little secondary structure or tertiary structure. Because of this, it does not denature easily. It is for this reason I expect that although the rate of reaction has dramatically decreased, it has not completely stopped. It could also be assumed that the enzyme has undergone the most denaturation because the substrate is less likely to denature.

The graph representing the rate of reaction (see analysis) throughout the course of this experiment, shows a definite curve. This is because at lower and higher temperatures the rate of reaction is much slower than at medium temperatures, as explained above. There is great difference between the rate of reaction at the highest two and lowest two temperatures. This shows that the activity of the molecules within the solutions respond the most to between the temperature changes of 20°C and 30°C, and 50°C and 60°C. This shows that between the temperature range of 30°C to 50°C, the body has adapted to be able to withstand a these temperatures and is also still able to function relatively well. Because there is a much smaller change in the enzyme activity rate between 30°C and 40°C (0.04) and 40°C and 50°C (0.02) it is obvious that the enzyme is reaching its optimum temperature. The body is able to withstand this range of temperatures so that it would not be fatally affected by, the more common, environmental extremes that would influence the internal body temperature.

Evaluation

The graph displays that as the temperature increases up to 40°C there is an increase in the rate of reaction. A greater increase in the rate of reaction is shown between the temperatures of 20°C and 30°C. After 40°C the rate of reaction begins to decrease, as the molecules are becoming denatured. The greatest reduction in the rate of reaction is between 50°C and 60°C. This is what I had predicted in my hypothesis, as discussed in the analysis. The plotted points on the graph form a curve. I have plotted a line of best fit, because I felt the results were reliable enough. This is because the readings and repeats measuring the absorbance at each temperature are very similar.

Anomalous Results

Although there are not any obvious anomalies, the range of the absorbance values fluctuated more as the temperature increased such as at 60°C when there is a difference of 0.10a between the highest and lowest result. This is because at higher temperatures, the solution was more affected when the solution was transferred from the water bath to the colorimeter as it cooled at a greater rate, as the rate of evaporation was greater. It was also more difficult to maintain the same temperature in the water bath at high temperatures because room temperature was so cool in comparison. Because the water molecules have more kinetic energy, they are vigorously vibrating and colliding with other water molecules, that as a result they are evaporating faster than usual in the form of steam, and the bath is cooling quicker.

The experiment was executed to the best of my abilities and all possible aspects to maintain a good operating procedure were done to gain accurate results. However, there were unseen problems in the experiments method and the apparatus used.

When pouring out the enzyme from the test tube into the test tube containing the substrate, it is possible that some of the trypsin solution may have been left behind if it adhered to the sides of the tube. This would affect the results, as different amounts would be left each time, therefore affecting the volume and concentration being used. This problem could be helped by adding a specific volume of the trypsin solution using a syringe, as the rubber seal located within the syringe mechanism would prevent as much being left behind.
When timing the reaction for exactly 120 seconds, it was difficult to transfer the solution into the colorimeter and measure the absorbance. As the transfer and reading took time, up to 15 seconds may have been added to the reaction time on some solutions and as little as 5 seconds on others. This would greatly affect the final result as the enzyme would have more time than the designated 120 seconds to break down the substrate. Although this is difficult to overcome, I could perhaps designate 10 seconds for putting the solution sample into the colorimeter per each solution. This would make sure there was continuity between each measurement time, but would still not solve the fact that the solution may cool at different rates within this 10 second time period.
Between each part of the experiment, the apparatus was washed, to prevent contamination. The syringes could not be dried adequately however and therefore some water may have been left inside. This may have diluted the solution and affected the results. This problem would be overcome by allowing the apparatus to thoroughly air dry after each wash which may be timely, or alternatively a different set of the same apparatus could be used for each batch.
During the experiment it is possible that, due to the busy laboratory environment I was working in, the various solutions could have been moved or jogged by other individuals. This would agitate the solution and increase the possibility of collisions between the enzyme and substrate, resulting in unfair results. If I was to conduct the experiment again, I would make sure I conducted the experiment when there was as little background movement as possible.
I also noticed that the measurements of rate of reaction became more variable at higher temperatures. This could be due to the difficulty in maintaining the water bath at a constant temperature. This is particularly true at high temperatures, when the solution will cool more quickly when transferring it to the colorimeter. If the experiment was conducted within an advanced colorimeter, this problem could be overcome.
The water bath maintained a temperature to within ± 0.5ºC and a thermometer was used to ensure the correct temperature. However, it was often difficult to maintain the exact temperature throughout the duration of the experiment, as it fluctuated. This may have resulted in an undesired change in temperature from one solution to the next. This problem would be helped with a more sophisticated water bath.
In my experimental method I did not control the pH of the reaction mixture. pH affects the activity of enzymes, and the pH of the mixture may have changed at different temperatures. Although there was little reason for dramatic changes in pH, I could have ensured a fairer test by adding a set volume of a buffer solution to the enzyme. Buffer solutions are used to keep the pH constant. I know from my background reading that trypsin works well at neutral to slightly alkaline pH, so a buffer with a pH between 7 and 8 would be suitable.
When the trypsin solution was added to the marvel milk solution, there is a possibility the temperature may have declined slightly when it was out of the water bath, especially at higher temperatures, this again would make the results less reliable.
When the trypsin solution was added to the marvel milk solution, the top of the joint solution was not covered by the water in the water bath. As a result, the solution at the top would have cooled and affected the experiment. This problem was made more apparent because when taking the sample from the solution to be measure in the colorimeter, it was taken from the top. If conducting this experiment again, I would use smaller quantities to ensure all the solution is covered, without having to fill the water bath up to more dangerously high levels, where it was obvious that it may overflow. Alternatively, I could use test tubes with a greater diameter as this would allow a greater volume of liquid to be inserted before reaching the top.
The experiment was carried out over two days. Environmental changes could have affected the experiment. An example of this is when measuring the solution sample in the colorimeter. As room temperature fluctuated, the reaction could have, for example, increased as it was being measured – especially when the experiment was being carried out at 20°, or slow down – especially at high temperatures such as 70°C. If done again, I would aim to start and complete the experiment using the same batch of solutions on the same day, within as short a time span as I could.
Two batches of the trypsin solution and marvel milk solution were used over the two days. Although a top hand balance and measuring cylinder were used both times, it was possible that the amounts measured out were different either time. The top hand balance is precise to two decimal places, so there was a possibility that, for example the first batch may have weighed less than the second batch. When measuring out the liquids, I used a measuring cylinder. This was measured in an accurate measuring cylinder, but human judgment may have influenced the point at which the quantity seemed correct. As above, if done again, I would aim to start and complete the experiment using the same batch of solutions on the same day, within as short a time span as I could.
Human reflexes may have influenced the point at which the experimenter saw it was time to transfer the solution into the colorimeter. As I was the only person conducting the experiment there is less of a chance that my judgment greatly varied from one solution to the next, but again conducting the experiment within the colorimeter, perhaps with a computer automatically taking readings at certain time intervals, this problem could be overcome.
The temperature increased each time by 10°C i.e. 20°C, 30°C, 40°C etc. When looking at the results it appeared that, as expected, the optimum temperature is 40°C. However, there is a possibility that the optimum temperature was ± 10°C of this value, as the optimum temperature could, in reality be as little as 35°C or as much as 44°C, for example. If I were to expand the experiment, this problem would provide the basis for it. I would carry out the same experiment with the above changes, but conduct them at 1°C intervals from 35°C to 45°C to discover, the more exact, optimum temperature.

Taking these issues into account it appears that although I did get the required results there were many changes that could have taken place to provide me with a fairer outcome. Although the repeats are similar at lower temperatures, fluctuating between 0.01A at 20°C, the range of the absorbance values fluctuated more as the temperature increased. An example of this is at 60°C when there is a total difference of 0.10A between the highest and lowest result. This is because at higher temperatures, the solution was more affected when the solution was transferred from the water bath to the colorimeter as it cooled at a greater rate, as the rate of evaporation was greater. It was also more difficult to maintain the same temperature in the water bath at high temperatures because room temperature was so cool in comparison. Because the water molecules have more kinetic energy, they are vigorously vibrating and colliding with other water molecules, that as a result they are evaporating faster than usual in the form of steam, and the bath is cooling quicker. Overall, the results plotted on the graph were as expected and I did not have any unexplained anomalous results, it is fair to conclude that my results are reliable.

References

(1) Cambridge Advanced Sciences – OCR Biology Specification A

(2) Cambridge Advanced Sciences – OCR Biology Specification A

Bibliography

Cambridge Advanced Sciences – OCR Biology Specification A
Problems in Practical Advanced Level Biology – P.W. Freeland
Biology Advanced Sciences – Tall and Tall
Oxford Encyclopedia - 2000 Edition
Revise As Biology for OCR – Richard Fosbery, Jennifer Gregory and Ianto Stevens.
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