To investigate how temperature affects the rate of reaction of the enzyme catalase on its substrate hydrogen peroxide

Back ground information:

Introduction: Enzymes are biological catalysts. They speed up metabolic reactions in the body but remain chemically unchanged themselves. Enzymes contain an active site. Enzymes are biological catalysts which increase the rate of reactions by lowering the activation energy needed for the reaction to take place. The activation energy is the amount of energy needed for molecules to react when they collide. Molecules need to collide in order to react, this is known as the collision theory. When they collide they may not react as a certain amount of energy is required to break bonds, (Hydrogen bonds, Ionic bonds and Disulphide bridges) this energy is the activation energy. The active site is a region, where normally another molecule may bind. This molecule is known as the substrate, and is usually specific to the active site of the particular enzyme, which breaks it down. Substrates will not usually fit into any other active sites other than that of the enzyme it is specified to. This can be explained as a lock and key hypothesis, where the lock and key are specific to each other, only, that there are many of the same kinds of lock and key when it come to the enzymes.

Just as lock and keys have three-dimensional shapes, proteins are also three-dimensional. Usually, there is only one active site on an enzyme; however there can be more. Some energy releasing reactions in cells produce hydrogen peroxide. This is acidic, and can thus, kill cells (denaturation).

Catalase is found in all living cells. It is used to remove hydrogen peroxide, which is produced during metabolism and is toxic if allowed to build up within cells. Celery cells contain the enzyme catalase which will be used as the enzyme in the experiment I carry out. Normally, hydrogen peroxide decomposes to form hydrogen (H2O) and oxygen:

2H2O2 2H2O + O2

Hydrogen Peroxide Water and Oxygen

In order to investigate the effect of temperature on the activity of catalase, I will record the amount of oxygen released when hydrogen peroxide is broken down.

Independent Variables (IV): There are quite a few independent variables which can alter the rate of reaction, and need to be kept constant. This is to ensure that the investigation is fair They are as follows:

pH: at too high pH, the enzyme is denatured due to the loss of tertiary structure (hydrogen & ionic bonds, disulphide bridges). This is because it distorts the bonds present. So the substrate no longer fits the active site. The same applies for too low a pH level. The active site will no longer be complimentary so the induced fit hypothesis will no longer be applicable The way in which I intend to overcome this problem is by using the same bottle of hydrogen peroxide throughout the experiment. This will ensure that the pH is kept constant.
Concentration of enzyme: The higher the concentration, the higher the rate of reaction will be. With a larger number of catalase molecules, the chance of successful collisions between enzyme and substrate will be increased. In order to keep this constant, I will make sure I use the same volume of celery (weight) containing catalase each time I conduct the experiment. I will keep the substrate concentration the same by using the same amount (weight) of celery, and using celery form the same bag if necessary.
Mass of Celery/Surface Area: Here it needs to be taken into account that different celery will give the same mass, if weight accurately. Even if accurately weighed different celery will not have exactly the same water and catalase content. . If surface area is altered there will be more enzyme active sites available in the space provided. Therefore more substrate can fit into those active sites, which will mean the rate if reaction will be altered
Incubation time: I will keep the incubation time for each individual experiment the same. This is in order to keep the experiment as fair as possible

The Independent variable (IV) will be temperature. The Dependent Variable (DV) will be pH, concentration of enzyme, mass of celery/surface area, incubation time and inhibitors.

Prediction

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The Independent variable (IV) will be temperature. The Dependent Variable (DV) will be pH, concentration of enzyme, mass of celery/surface area, incubation time and inhibitors.

Prediction

The higher the temperature, the higher the rate of reaction up to a certain point. I think that up to 40°C the reaction will be fast. After this temperature is reached the enzymes will denature. This is due to the fact that the particles gain kinetic energy and subsequently move around more vigorously (vibrate more). Thus, the chance of there being a successful collision between the enzyme and substrate molecule increases as reacting particles with collide more frequently with increased kinetic energy. I predict that at 60°C there will be no reactions taking place hence no gas being produced. This is because all of the active sites will be denatured due to the high temperature. This is because the enzyme catalase is not thermostable.

Method

As oxygen is produced in the reaction it will displace the air trapped in the conical flask, this will be forced through the delivery tube into the measuring cylinder. The air will rise to the top as the measuring cylinder is full of water and its volume can be measured. Take readings of gas produced every 1 minute for five minutes.

Other methods which can be used are counting the number of bubbles produced, this would not be accurate as the bubble sizes are not the same and the volume is not measured. Measuring the mass of the gas lost is a better method as readings on the scale will be accurate, however this would require equipment which is not available. Another possibility could be measuring the height of the bubbles. But this would be unpractical because the bubbles being produced would be constantly bursting, which would have an dramatic effect on my results.

Apparatus required

1 piece of celery (medium sized)
Measuring cylinders (100 cm3 and 50cm3 )
Hydrogen peroxide solution (H2O2)
Water (H2O)
Ruler
Magnifying glass
Blender
Stop watch
Clamp stand Boss
Bung & Tube
Large plastic bowl
Conical flask (with rubber bug attached)
Long rubber tube
Weighting scales (to .2 decimals accurate)
Small beaker
8 Water bath’s (set at different temperatures)
Thermometer (0°C - 100°C)
Goggles
Apron

The experiment will be safe by:

Always wearing safety goggles, as hydrogen peroxide an damage your eyes,
Not spilling the hydrogen peroxide as it is an irritant, and bleaches,
Taking care when using the blender to blend the celery into a paste,
Taking care when handling hot water.

The temperature of celery I am going to in my investigations will be as follows: 20ºC, 30ºC, 35ºC, 40ºC, 45ºC, 50ºC, 55ºC, 60ºC. This is so I can get a good range of results and an average as I will be doing each experiment 3 times.

First of all I had to set up the apparatus. After this is done , I need to set up the blender and place a piece of celery into the blender. Once I have done this, I need to turn on the blender on and blend the celery until it turns into a paste. Once this has been blended, set up the weighting scales and place an small empty beaker onto of the scales, making sure the beaker is empty and clean of any debris. One placed onto the scale press the reset button and pour in the celery which has been blended into a paste and measure 5.0g accurately. The reason for pressing the reset button is so that only the celery is weighted not the celery and the beaker. Also to make sure that the experiment is as fair as possible. Once weighted carefully place into a conical flask, making sure all off the content is transferred from the beaker to the conical flask. Once this is done place the conical flask in the water bath at the desired temperature. This is to ensure all of the celery is at the same/desired temperature. If this was not done then the test would have not been fair, because of the celery not being at the temperature I had chosen. This would effect the results because it would take longer for the reactions to start because the activation energy (Ea) take longer to reach.

As the celery is reaching the desired temperature, I will be measuring out 10 cm³ of Hydrogen Peroxide (H2O2). When I am measuring the Hydrogen Peroxide the readings should be taken with the graduated markings at eye level, so that the measurements are accurate. Once measured accurately, place the Hydrogen Peroxide in the same water bath as the celery (Catalase). Once this is completed, place a thermometer into the water bath so the temperature can be checked and kept constant accurately. As the celery and the Hydrogen peroxide are reaching the desired temperature, set up the next part of the experiment. get a large plastic bowl and fill with water (hot or cold). Then get the small conical flask and fill with water, so there are no air bubbles present (see diagram). If there were to be any air bubbles present this could seriously effect the results. Once the conical flask is filled with water place it at the bottom of the plastic bowl full. Nest check the temperature of the celery and Hydrogen Peroxide. If they are at the desired temperature then you are ready to proceed to the next step.

Get the bung/conical flask and the delivery tube ready. Place the delivery tube under the measuring cylinder (see diagram) then get the celery and pour into the conical flask with the bung and delivery tube attached to it. Once all of the celery is added place the conical flask into the water bath to maintain the desired temperature and to make the test as fair as possible. Then place the Hydrogen Peroxide into the same conical flask and simultaneously start the stop watch and replace the bung on top of the conical flask. Record the volume of gas produced every 1 minute for 5 minutes in a well constructed table. When taking a reading make sure the readings are taken with the graduated markings at eye level, so you can take an accurate reading. Also when taking results down make sure that the rest of the Dependent Variables (DV) are kept constant, in order to make the test fair. Once 5 minutes have been, dispose of the celery (Catalase) and Hydrogen Peroxide. This is because all of the active sites will be used up or denatured (depending on temperature). Then repeat the experiment for each and every temperature, making sure that each and every apparatus is cleaned hence making the experiment fair.

Diagram of apparatus

A Table to show the Volume of Oxygen Produced due to the Catabolic breakdown of Hydrogen Peroxide in Relation to Temperature

Each experiment was carried out 3 times, so I could get a good set of raw data hence being 3 tables.

Set of results 1

Set of results 2

Set of results 3

Averages

(all measurements in cm³ unless stated)

Analysis of results

The results obtained during the course of the experiment seem to be quite conclusive. It possible to identify a pattern or trend in the results obtained. From the graph, we can see that the oxygen is produced more and more quickly via the breakdown of hydrogen peroxide with an increase in temperature. This is, however, only up to a certain point. We see that the rate of reaction keeps on increasing until 40ºC, after which it starts to fall. There is still some oxygen being produced after 60ºC, but only very little, near to zero. This happens because at low temperatures the hydrogen peroxide has less energy, so moves more slowly. It will collide with the catalase less often, meaning the frequency of collisions is low. They are less likely to have the right activation energy so there are less collisions resulting in reactions. This will mean the rate of reaction will be low. At higher temperatures the hydrogen peroxide has enough energy to reach its activation energy and it is also colliding more often, so the rate of reaction will be higher.

After 40°C the rate of reaction falls , this is because the catalase begins to denature (loss of tertiary structure). When enzymes denature the attractions between amino acids in the enzyme break and the enzyme begins to return to its original shape. The shape of the active site also changes so it cannot break down the hydrogen peroxide

With respect to the results obtained, I can now say that the prediction I made earlier on was more or less correct, although not as correct as I had hoped they would be, as I had predicted that the rate of reaction would be zero after 60ºC. However, the rest of my prediction seems to be in support of my hypothesis. Thus, it would seem that the enzyme activity (rate of reaction) increases with temperature up until around 40ºC as the enzyme and substrate molecules gain more and more kinetic energy. As a result, the reactants move around with increased vigour. This results in there being an increased number of effective collisions. Subsequently, the rate of reaction increases. After 40ºC, the rate of reaction deteriorates. Although the kinetic energy increases, the rate of reaction should keep on increasing, this is however not true. This due to the fact that after 40ºC, the optimum temperature for catalase enzyme activity, the weak bonds that hold together the enzyme structure, start to break (this is especially true of hydrogen bonds), due to the increased kinetic energy. Resultantly, the rate of reaction deteriorates as the enzyme becomes denatured due to the fact that its active site, and ultimately its whole structure, is lost due to the breaking up of the bonds that hold it together. This means that the substrate molecule can no longer fit into the active site of the enzyme as the shape of the active site changes.

Evaluation

Although the results obtained from the experiment were more or less support in my prediction, I was not totally satisfied with the experiment. There were a lot of errors, both in the conducting of the experiment, and in the results obtained.

First of all the way I conducted the experiment was not very good. For example, the measuring cylinder used to measure the volume of oxygen produced, was quite inappropriate, and probably resulted in many of the readings being taken being quite inaccurate as it is easy to make a mistake in reading off the value while trying to hold the measuring cylinder straight. Also, it was only possible to measure to the nearest 0.5 of a centimetre cubed, as the measuring cylinder was only graduated in 1cm3

The method used to keep the temperature of the reactants constant at certain temperatures was also improper, as water in the beaker which was used for the above purpose, was not in a large enough volume or quantity to retain its heat for more than a minute or two. Thus, the temperature of the reactants was fluctuating during the experiment. Also, the water in the beaker did not cover all the reactants in the conical flask, and some of the reactants were not immersed by water. Thus, there were regions of unequal temperatures in the reactants.

Also, it was assumed that the pH of the reactants would remain constant throughout. This may not have been the case.

Considering the above, it is feasible to say that the results obtained during the experiment are neither likely to be very reliable nor very accurate.

Considering that the there was so much possibility for inaccuracy, there were not however, any major anomalies in the results obtained.

These slight anomalies may have arisen may have arisen due to a number of reasons:

a) Improper measurement off measuring cylinder

b) Improper surface area of celery, and effectively enzyme

c) Difference in temperature due to loss of heat (kinetic energy)

d) pH level may have altered

If I was to conduct the experiment again, I would make sure that it was more accurate overall. This would ensure that the results obtained were more reliable and accurate. I would do this in the following ways:

1) I would use a graduated syringe, instead of a measuring cylinder as this would ensure that it was easier to measure the volume of oxygen produced. It would also be more accurate as there would be a clear line to mark the amount of oxygen produced.

2) I would use a greater volume of water to ensure that the temperature of the reactants remained constant, due to the fact that water is such that, the greater the volume, the greater its ability to retain heat.

3) I would use a buffer to control the pH of the reactants as it would ensure that the pH remained constant.

4) I would also use a graduated syringe to measure out the amounts of hydrogen peroxide, and other fluids. The reasons for this are the same as the ones mentioned above.

With the above taken into account, I would say that my conclusion is in fact, not very safe due to the fact that there were too many errors and uncertainties concerning the results obtained. Subsequently, the results do not provide a very stable evidence for support of my hypothesis. Although the percentage error of individual equipment may have been at first glance, small, they add up to large percentage errors which then render useless, the legitimacy of the results obtained