The Effect of Temperature on the Rate of an Enzyme Catalysed Reaction

Aim: To find the effect of temperature on enzymes, using a potato as a catalyst. The source of catalase is in the potato cells.

2H2O2 → O2 + H2O

Planning:

Introduction: An Enzyme is any one of many specialised organic substances, composed of polymers of amino acids, that act as catalysts to regulate the speed of the many chemical reactions involved in the metabolism of living organisms

Enzymes are classified into several broad categories, such as hydrolytic, oxidising, and reducing, depending on the type of reaction they control. Hydrolytic enzymes accelerate reactions in which a substance is broken down into simpler compounds through reaction with water molecules. 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.

Individual enzymes are named by adding 'ASE' to the name of the substrate with which they react. The enzyme that controls urea decomposition is called urease; those that control protein hydrolyses are known as proteinases. However, some enzymes, such as the proteinases trypsin and pepsin, retain the names used before this nomenclature was adopted

Enzymes are large proteins that speed up chemical reactions. In their globular structure, one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site, or the location on the enzyme where the substrate binds and the reaction takes place. Enzyme and substrate fail to bind if their shapes do not match exactly. This ensures that the enzyme does not participate in the wrong reaction. The enzyme itself is unaffected by the reaction. When the products have been released, the enzyme is ready to bind with a new substrate

Properties of Enzymes

enzymes are typical catalysts: they are capable of increasing the rate of reaction without being consumed in the process.

Some enzymes, such as pepsin and trypsin, which bring about the digestion of meat, control many different reactions, whereas others, such as urease, are extremely specific and may accelerate only one reaction. Still others release energy to make the heart beat and the lungs expand and contract. Many facilitate 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.

enzymes are very efficient. Small quantities of an enzyme can work at low temperatures what would require violent reagents and high temperatures in ordinary chemical reactions.

Method

I will be looking at the effect of catalase (in potatoes) on Hydrogen Peroxide to find the effect of temperature on the rate of an enzyme-controlled reaction. Oxygen gas will be produced therefore we can measure the volume of oxygen produced, and compare the rate which is produced at different temperatures to see the effect that changing temperature has on the rate of reaction. I will obtain my results by measuring oxygen evolution in cm3 over various temperatures. If graphs are produced we can measure the gradients of the graphs and we will obtain values for the rates of reaction, which can then be plotted on a separate graph.

I will use a range of temperatures of 10, 20 30, 40, 50, 60 and 70oC to give me a wide range of results which will help me find the results for my task. Any anomalous results should be repeated to check that they are correct. Hopefully the experiment shall be repeated 3 times at least so that I can obtain an average set of results to minimise the effect of any uncontrolled variables.

H2O2 is a good substrate to use because we know exactly how much oxygen gas should be produced. We should use Hydrogen Peroxide from the same batch so that it will decompose the same amount. It should be stored in cool conditions to minimise decomposition. I am investigating the effect of changing temperature on a reaction. If this is to be a fair test with reliable useful results obtained other variable factors need to be controlled. Enzyme controlled reactions are very sensitive to many factors and are easily affected. I want to keep these other factors constant throughout our tests whist we only vary temperature so we can see what affect changing this factor alone. I have chosen to use potato in our experiment because the catalase enzyme is present in potato and we can also easily control the surface area and quantity of potato, which is important.

When temperature is increased the increased heat energy raises the kinetic energy of the reacting molecules leading to more frequent collisions between substrate and enzyme. More enzyme substrate complexes are formed per second, with a corresponding increase in the rate of catalysis and on the number of product molecules. Therefore the temperature of the environment surrounding the system should be kept constant and the temperatures variables should be accurate.

I already know that concentration affects enzyme activity as when concentration is increased there are more active sites available for reaction so collisions occur more frequently between substrate and enzyme, so there are more substrate complexes which form per second- so the rate of reaction increases. If enzyme concentration is varied then this will affect the rate of reaction. Can attempt to control this by using the same potato for each experiment. Surface are should be kept constant. If the piece of potato is for example cut into smaller pieces whilst another is left in a larger piece it will have a greater surface area If the potato has a larger surface area then there will be a much larger area upon which the collisions between substrate and enzymes occur. More enzyme substrate complexes will form per second and the rate of reaction will increase. To control surface area we need to use a potato borer to keep surface are constant throughout. It is also important that we use the same potato for each experiment as different potatoes could have different concentrations of enzymes within them and the y could also be of different ages, which could affect the rate of reaction

These are all the variables that I feel need to be controlled within the experiment so that the rate of reaction is not affected by anything other than temperature. However I also need to record my results at regular intervals so I can fairly compare the rates of reactions between different temperatures. I will take the readings of the volumes of oxygen produced every 20 seconds from immediately after the Hydrogen Peroxide has been added to the conical flask containing the potato. It is important that results are obtained at the same frequency so that we can see how oxygen evolution occurs across the whole experiment.

Apparatus

Bench mat

Goggles (for safety)

Conical Flask

Graduated pipette

Bung and delivery tube

Syringe

10 cm3 Hydrogen Peroxide

Boss and clamp

Stand

Gas syringe

Ice

Kettle

Potato

Borer

Stopwatch

50 ml beaker

2*250ml beaker

Thermometer

Use a size 5-potato borer to extract a length of potato. Cut this equally into 10 pieces of length 5mm. Put these pieces into a conical flask. Dispose of the excess.

Pour around 25cm3 of Hydrogen Peroxide into the 50 ml beaker.

Pour water into both 250ml beakers and cool them to 10 oC. Check with a thermometer. Put the conical flask in one beaker and the smaller beaker into the other. Make sure they do not sink. Leave them to adjust to the temperature and constantly check the temperature to make sure it is right.

Using the graduated pipette, 10cm3 of Hydrogen Peroxide should be obtained and placed into the syringe.

Check that the gas syringe is set at 0

Apply pressure to the syringe containing the Hydrogen Peroxide until all the contents have been added to the conical flask.

Immediately start the stopwatch. Take readings of oxygen evolution every 30 seconds and record them in a table. After 240 seconds stop recording results.

Wash thoroughly all apparatus and repeat the above steps using the different temperatures of 20, 30 40 50, 60 and 70oC. For the higher temperatures I will be placing in a water bath.

I am going to repeat my experiment so that I have 2 sets of results

I shall only be altering the temperature, as that is my main variable, and so I will therefore keep the others the same in order to make it a fair test. I will keep my variables constant by making sure that I have used the same technique for each experiment. The amount of Hydrogen Peroxide will be carefully measured using ameasuring cylinder; enable me me to make sure it is kept constant throughout the experiment.

Safety Precautions: I will have to be careful when using the Hydrogen Peroxide, as it is a corrosive chemical, so I will attempt to overcome this problem by wearing goggles. Hydrogen Peroxide is a bleaching agent, so I will be wearing a lab coat so it does not bleach my clothes. I will also be using sharp razors during the experiment so I will have to be cautious about that so tht I don't end up cutting myself.

Hypothesis: My prediction is supported by Kinetic Theory in that if I apply twice as much heat there will be twice as much particle vibration therefore the reaction will happen twice as quickly. It is also backed by Collision Theory in that if I apply twice as much heat there will be twice as many collisions and therefore the rate of reaction will double. This will only be so until the enzyme denatures after its optimum temperature: 40 - 45°C

Predictions: I predict that the enzyme will become denatured, and therefore will work at a slower rate after 40 - 45°C. I think the reason for this prediction is because every enzyme has a temperature range of optimum activity. Outside that temperature range the enzyme is rendered inactive. This occurs because as the temperature changes enough energy is supplied to break some of the molecular bonds. When these forces are disturbed and changed the active site becomes altered in its ability to accommodate the substrate molecules it was intended to catalase. Most enzymes in a human body shut down beyond certain temperatures. This can happen if body temperature gets too low (hypothermia), or too high (hypothermia).

Analysis

From the graph, I am able to back up my theory. I can see when the enzyme is most active and when it starts to denature. From the graph, I have found out that, as the temperature increases, so does the catalase activity, as it does not take as long to move the same distance, up to a certain point ( °C) where the activity ceases altogether. I found that the optimum for a catalase is at °C. This is where the greatest number of collisions takes place between the enzyme and the substrate and therefore the highest rate of reaction is.