An Investigation into the Effects of Temperature on Enzyme Reactions

Objective- To find the effect temperature has on the reaction between Trypsin and Albumin.

Introduction

An Enzyme is a specialised organic substance, composed of polymers of amino acids that act as catalysts to regulate the speed of many different chemical reactions involved in the metabolism of living organisms. There are more than 700 different enzymes.
They can be classified into several categories, such as hydrolytic, oxidising, and reducing, depending on the type of reaction they control or influence. Hydrolytic enzymes use water molecules in the break down of substances into smaller substances. Oxidising enzymes, known as oxidises, accelerate oxidation reactions; reducing enzymes speed up reduction reactions, in which oxygen is removed. Many other enzymes catalyse other types of reactions.
The Lock and Key Theory:

In their structure, one or more polypeptide chains change, bringing together small numbers of amino acids to form the active site- the location on the enzyme where the substrate binds and the reaction takes place. If the enzyme and substrate fail to bind (their shapes do not match exactly), a reaction fails to take place. This ensures that the enzyme does not participate in the wrong reaction. The enzyme itself is unaffected by the reaction only the substrate. When the products of the reaction have been released, the enzyme is ready to bind with a new substrate.

Trypsin, which is secreted by the pancreas and brings about the digestion of meat, controls many different reactions, whereas others, such as urease, are extremely specific and may accelerate only one reaction. Others release energy to make the heart beat and the lungs expand and contract. Many aid the conversion of sugar and foods into the various substances the body requires for tissue-building, the replacement of blood cells, and the release of chemical energy to move muscles.
Trypsin and some other enzymes possess, in addition, the peculiar property known as auto catalysis, which permits them to cause their own formation from an inert precursor called zymogen. As a consequence, these enzymes may be reproduced in a test tube.
As a class, enzymes are extraordinarily efficient. Very little quantities of an enzyme can achieve a low temperature reaction, which would usually require violent reagents and high temperatures. The kinetics of enzyme reactions differs somewhat from those of simple inorganic reactions. Each enzyme is selectively specific for the substance in which it causes a reaction and is most effective at a temperature and pH peculiar to it. Although an increase in temperature may accelerate a reaction, enzymes are unstable when heated. Whereas an increase or decrease in the pH would denature or destroy the enzyme. As a rule, enzymes do not attack living cells. As soon as a cell dies, however, enzymes that break down protein rapidly digest it. The resistance of the living cell is due to the enzyme’s inability to pass through the membrane of the cell as long as the cell lives. When the cell dies, its membrane becomes permeable, and the enzyme can then enter the cell and destroy the protein within it. Some cells also contain enzyme inhibitors, known as anti-enzymes, which prevent the action of an enzyme upon a substrate.

The Albumin hopefully does not contain any anti- enzymes that will prevent a reaction between it and the Trypsin. This is what must be found in the preliminary experiment.

Preliminary Work

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Preliminary Work

A preliminary experiment was performed to test that there was actually a reaction between the Trypsin and the Albumin. The same equipment that was intended to be use for the actual experiment was used (see page 4). The water in the water bath was heated to an estimated peak temperature of 38°c and a test tube containing 2ml of Trypsin and 2ml of Albumin was placed into the water bath. A stopwatch was started at this time. This was to check how long it took the Trypsin to break down the Albumin. This gives a good basis for how long to leave the experiment to finish. After a certain time the colour of the solution began to become clearer. This shows that Albumin does not contain an anti- enzyme and that Trypsin breaks down the Albumin so these two substances are good for an experiment.

From the research shown in the introduction the pH that Trypsin works best in is pH8. Works best means it breaks down particles quicker and more efficiently. This can be achieved in this experiment by using a pH8 buffer to keep the solution at pH8. Knowing the solutions needed the ratio of how much of each must be created. A 1 : 2 ratio can be used, because this would give enough Albumin for the Trypsin to break down and give enough Trypsin to do it. But this still leaves the buffer. As the buffer only needs to keep pH it can be added as 1 : 2 : 1. Where 1 is the Trypsin and the buffer and the 2 is the Albumin. There is the same amount of buffer as there is Trypsin but more Albumin. This ratio should give a good enough imitation of the body. The only factor being left out is temperature. Temperature was not tested in the preliminary experiment because it is the main variable for the experiment.

The results of the Preliminary Experiment:

The pH of the solution must be kept at pH8 using a buffer.
The ratio of Trypsin: Albumin is 1 : 2
The ratio of Trypsin: Albumin: Buffer is 1 : 2 : 1

Also the actual volumes used will be 1 : 2 : 1 e.g. 1ml : 2ml : 1ml.

Variables

Temperature- is the independent variable. This will be controlled and set to required levels to give accurate and adequate results.

Vol. of Trypsin – is constant variable because it remains constant throughout the experiment. If this changed then the results would not be fair i.e. if there are more enzymes then there are going to be more reactions and therefore in the 5minutes given for the experiment to take place, the solution will be clearer then if there had been the same amount of Trypsin all the way through.

Vol. of Albumin- is a Constant Variable. If there ridiculously more albumin in one experiment compared to another then there will be more substrate particles o break down and there will not be enough Trypsin to break down as many in the 5 minutes because they will loose energy. This is why a constant amount of Albumin will keep the Trypsin working at the same rate as all the other experiments.

Concentration of the Two Substances- is constant. If one is only 50% of what it should be then it is only going to have 50% of the reactions take place.

pH of Solution- is constant because if this was more acidic or more alkali then the enzyme will not work as well as it work in its own pH environment. If this changed then the enzyme activity would change and influence the results.

Hypothesis

If this experiment goes to plan and is fair, I believe that when the temperature is high the enzyme- trypsin, will not work very well and will not break down the Albumin as well as it will at lower temperatures. This is because it will denature and its active site will no longer be able to bind with the substrate. Around 30°- 40° is where I think there will be the highest breakdown of Albumin and even lower than this these temperatures I think will not give the enzyme enough energy to work very well. I think that this experiment will produce very similar graphs to those that follow the enzyme theory. A rise to a peak at about 37’ and then a drop, where it begins to denature. This will only happen if the experiment is performed correctly.

Equipment

Trypsin- the main enzyme.
Albumin- the substrate which will be broken down by the enzyme.
Colorimeter- to accurately read the colour absorption and to accurately see how much Albumin has been broken down.
Test Tube(s) - number will depend on number of temperatures and repeats needed for accurate results.
Bunsen- to heat water and keep at constant heat.
Beaker(s) - to act as water bath and then test tube with experiment.
Thermometer- to check the temperature of the solution and the water.
Tripod and Gauze- to carry the water bath.
H2O- for heating.
Pipette to accurately measure the amount if solutions being used.
pH8 Buffer- to keep pH of solution to pH8.
Measuring Cylinder- to measure amounts of solutions.

This equipment will ensure a fair and safe test providing accurate results.

Diagram

Method

The temperatures used should be 0°c, 10°c, 20°c, etc up to 60°c. This gives a wide range of results and will allow us to find the peak of the enzyme and where it begins to denature, if it does at all.

Gather all equipment and ensure they are they are a clean. Set-up as shown in diagram.
Place 50ml of water in a beaker and begin heating to required temperature, checking with thermometer.
Place 1ml of Trypsin, 2ml of Albumin and 1ml of Buffer into a colour vessel and take a reading. Repeat this three times and take an average. Note this reading and name it your CONTROL.
When temperature is at required level remove Bunsen but occasionally replace to maintain the temperature. Place a test tube in to the water bath and leave for about a minute for it to achieve the same temperature as the water.
Take the test tube out of the water and place appropriate amounts of Trypsin, Albumin and Buffer into the test tube and place back into the water bath.
At this point a stopwatch should be started and the experiment should be left for 5 minutes.
Once 5 minutes is over take the test tube out from the water and pour some of the solution into to a colour vessel and test for colour absorption in the colorimeter. Take down these readings in a table.
Repeat steps 4 – 7 with all the other temperatures required.
Then repeat steps 4 -8 a further two time to gain averages and accurate results.

SAFETY: Normal rules of the Lab.

Results

A colorimeter measures solutions in ABS. It measures the solutions by how much light they absorb i.e. not let through to the detector on the other side of the colorimeter vessel.

This reading gets lower as the solution becomes clearer e.g. a clear solution would be around 0.00 ABS and a dark solution would be around 50.00 ABS.

AS we can see from the graph, the reading is high at the very low temperatures, this is where the enzymes do not have enough energy to move around and collide with the substrate. As the temperature begins to get higher, the readings drop quite suddenly. This is where the enzymes have enough energy and are moving around quickly and therefore the are breaking down the substrate quicker and more often. This is happening at around 20-30°c. After this period the readings begin to rise again and then continue to rise steadily. This is because there is much more heat around in the solution and the enzymes begin to denature. This means that the substrate can no longer fit in to the enzyme and is not broken down and therefore e the solution remains milky.

Within my results there is only one odd results, which is the 20°c reading. This sample broke down more of the substrate considering the temperature.

Conclusion

Looking at enzymes and Trypsin

The reason why reactions occur is because molecules or other particles are given enough energy to collide with each other at such a velocity that they bond together. This does not happen at low temperatures because the particles receive they energy from heat. As a solution becomes hotter the particles are given more energy and they react more quickly, until they are given too much, this causes the particles to collide with each other at such a velocity that when they collide they disfigure each other. In the case of enzymes, they become denatured.

Normal body temperature and pressure are too low for a chemical reaction to occur at a rate rapid enough to maintain life. Rising the temperature, pressure or even the number of reacting enzymes could denature proteins and damage or kill cells.

Enzymes are the solution to this problem. They speed up reactions by lowering their Ea and directing colliding molecules. The Ea is the activation energy and is the amount of energy needed to disrupt the stable electronic pattern of a specific molecule so that the electrons can be re- arranged. Even if the particles have enough energy to react they will not because they need to be oriented into each other to react. Enzymes do this without disrupting the body. These factors make enzymes biological catalysts. The structure of a enzyme can be very complex. They consist of a protein called the Apoenzyme and a non- protein portion called the cofactor or an organic molecule called a coenzyme. These are often vitamin derivatives. Together these parts make the Holoenzyme or the complete enzyme. The specificity o fan enzyme as has already been explained in my introduction is very important to an enzyme. Each enzyme can only catalyse one type of substrate. It is the active site of the enzyme in which this substrate enters and fixed to the enzyme if it is the correct shape for it to fit into (lock and key theory). However sometimes the active site changed to accommodate the substrate, this is called an “induced fit”. The main aim of an enzyme is to find its substrate and break it down in to the product. Also as stated in my introduction, enzymes are incredibly efficient they can catalyse reactions at rates millions the time faster than allowing the reaction to take place without them e.g. with tremendous heat. The number of substrate molecules that can be broken down in a second, ranges from 1- 600,000. The only way in which enzymes can do this is if they are controlled correctly. They are all controlled by the cells from which they were secreted. The concentration or the way in which they break up molecules is controlled by the cell. The enzyme in this experiment was Trypsin.

Trypsin breaks down proteins into peptides. It is secreted by the Pancreatic Acinar Cells in the Pancreas and is sent to the stomach to breakdown protein. However Trypsin breaks down proteins. Muscles are proteins, and the small intestine in which the pancreatic juice that contains the enzyme travels, is a muscle. Therefore the enzyme would begin to digest the wall if the inner small intestine. This is why the Pancreatic Acinar Cells secrete trypsin in the form of Trypsinogen. This is an inactive form of the enzyme that should not break down proteins. In case Trypsinogen is not produced correctly and instead become the active enzyme, Trypsin, the Pancreatic Acinar Cells also secrete a protein, called Trypsin Inhibitor which combined with the Trypsinogen to block it’s enzymatic activity. When the Trypsinogen and it’s inhibitor reach the end of the small intestine, they come into contact with an activating enzyme call Enterokinase which spits the parts of Trypsinogen and destroys them along with the Trypsin Inhibitor and leaves the active enzyme to break down protein in the stomach. In turn the Trypsin firstly breaks down the proteins inhibiting the other enzymes that are in the Pancreatic Juice.

It for these reasons that we can #tell that the form of the trypsin used in this experiment was not from the body. The Trypsin used was created from an inert precursor called Zymogen. Trypsin could be formed from this because it contains a property known as an auto-catalyst, which allows them to form like this. This does in no way mean that the experiment was unfair, however it does bring to our attention that this experiment was only a “simulation” of the body.

In conclusion, Enzymes are very complex parts of every day life and changes in temperature can cause them to act in very different ways that can be harmful to organisms.

The results and the conclusion support my Hypothesis and further prove it to be correct. Everything stated in my Hypothesis was true in the experiment and it described the graph almost exactly before it was drawn.

Evaluation

The data obtained in this experiment supports my conclusion well.

However there certain area in which I think there could have been better planning e.g. the odd results at 20°c- reading could have been taken at 5°c intervals instead of 10°. This would have given more results to plot on a graph and see if this was actually an anomaly or not.

I am convinced that the results show there is a correlation between the temperature and the Colour of the solution. The accuracy of these measurements could be improved by the use of a burette instead of a measuring cylinder, as it is a more precise piece of equipment and there are fewer margins for error. Another source of error may have been in the water baths, as they were supposed to be set at fixed temperatures to heat up the substances. One had to be very careful that the substances did not exceed or drop their planned temperatures or the experiment would not be fair. This could have been resolved by using an electronic water bath that would be fixed at temperature using a thermostat. The experiment in its entirety was successful, but it could be improved by the use of more accurate equipment and better organisation.