catalase
2H2O2 2H2O + O2
This equation reads that 2 molecules of hydrogen peroxide (the substrate), in the presence of the enzyme catalase, will decompose to form 2 molecules of water and one molecule of oxygen (the products). As stated above this reaction happens naturally anyway but the enzyme catalyses the reaction. This happens because the substrate possesses kinetic energy and is therefore randomly moving around and comes into contact with the enzyme’s active site. Here the enzyme forms temporary bonds with the substrate and therefore changes its shape, thus lowering its activation energy. The energy give to the substrate through increased temperatures in the body allow it to break down and thus the product leaves the enzyme. This is represented in the following diagram.
Liver tissue contains a large number of peroxisomes, as it is the main site of the breakdown of hydrogen peroxide. It therefore also contains a high concentration of catalase. However liver is difficult to manipulate and work with so in our experiment, celery tissue will be used. Celery tissue possesses catalase in the cytoplasm of all its cells. Because of this we can presume that the volume of the enzyme catalase is represented by the amount of celery tissue present as the enzyme is uniformly spread around the tissue.
Prediction:
My prediction of the relationship between the rate of the breakdown of hydrogen peroxide, catalysed by the enzyme catalase and the concentration of the enzyme is as follows: As the concentration of enzyme increases, the rate of reaction will also increase. The two variables, concentration and rate should be directly proportional, that is if the concentration is doubled, the rate of reaction will also double. This would be represented on a graph of concentration of enzyme against rate of reaction by a straight line through the origin. This direct proportionality will be true indefinitely if the amount of substrate is kept in excess. Otherwise it will be a limiting factor to the experiment as there will not be enough substrate available for the higher concentrations of enzyme, so the rates will not increase. When talking about the rates of reaction for different concentrations of enzyme, the initial rate of reaction must be considered. That is as the reaction is an ongoing thing; it is fairest to look at the rate at the beginning of the reaction. This is because as the reaction carries on, the amount of substrate present decreases and this reduces the rate of reaction, as explained below. Therefore the rates of reaction vary with each experiment and decrease by different amounts depending on the concentration of enzyme.
This relationship can be explained very simply by looking at how the reaction takes place. By increasing the concentration of enzyme, the amount of enzyme per a certain area is increased, or there are more enzymes present in a given area. In order to be catalysed, the substrate has to come into contact with the enzyme to join to its active site through its own random movement as well as the random movement of the enzyme (due to the kinetic energies of the two substances). By increasing the amount of enzymes present the amount of active sites present are increased and therefore the likelihood of the substrate joining to the active site is increased and thus the rate of reaction is increased. The relationship is a linear one because the amount of active sites is directly proportional to the likelihood of the substrate slotting into it and therefore the rate of reaction. Equally a decrease in concentration results in a decrease in the rate of reaction.
There are a number of other factors apart from concentration of enzyme, which affect the rate of an enzyme-controlled reaction. As we are investigating concentration of enzyme only, these other factors need to be controlled or at the very least taken in to account when drawing conclusions. This is necessary as if any other variables are changed, they could have an effect on the rate of reaction instead and the test will not be a fair one. The factors which need to be controlled or measured and taken into account are listed below.
- CONCENTRATION OF ENZYME- As stated above, the concentration of enzyme affects the rate of reaction because an increase in concentration means an increase in the number of active sites available to the substrate. Concentration of enzyme is the independent variable which when altered should have an effect on the dependant variable, rate of reaction. To vary the concentration of enzyme, the celery must be liquidised in a blender to form celery ‘extract’. As the enzyme is found in roughly the same amounts in every cell in the celery, the concentration of the celery ‘extract’ is a measurement of the concentration of enzyme. The concentration of enzyme can therefore be altered, by varying the concentration of celery ‘extract’. This is easily done by adding distilled water to form an ‘enzyme solution’ as the enzyme easily dissolves in water (due to its R group).
- VOLUME OF ENZYME- An increase in the volume of enzyme solution also increases the rate of reaction, as again there are more active sites present. When varying the concentration this must be taken into account and the overall volume of enzyme solution must be kept constant. This is done by measuring the concentration of enzyme as a percentage of celery extract in a fixed volume of enzyme solution. We assume that pure celery extract equals 100% concentration of enzyme. If the amount of celery extract in the fixed volume is halved and the other half of the solution is made up with distilled water, this is referred to as 50% concentration. Similarly, if the celery only makes up a quarter of the total solution (the remaining three quarters is made up by distilled water) this is 25% concentration. E.g. If the volume of solution is 10ml, 100% concentration would be 10ml of celery extract. 80% concentration would be 8ml of celery extract and 2ml of distilled water. 60% concentration would be 6ml of celery extract and 4ml distilled water …etc. This alters the concentration, without altering the volume of solution.
- CONENTRATION AND VOLUME OF SUBSTRATE- The concentrations and volumes of the substrate (hydrogen peroxide) will affect the rate of reaction. If more substrate is present, then there will be a greater chance of collision with the enzyme when it’s in solution. This means that there is a greater chance of the enzyme and substrate forming temporary bonds and the enzyme thus lowering its activation energy. So an increase in concentration of volume of substrate will increase the rate of reaction. The concentrations and volumes of hydrogen peroxide can easily be measured and must not only be kept constant, but must also be kept in excess. If there is not enough substrate available to the higher concentrations of enzyme, it will limit the rate of reaction. That is an increase in concentration of enzyme will have no effect on the rate of reaction because there will not be enough substrate for each enzyme to work on.
- TEMPERATURE- A rise in temperature is brought about by an increase in heat energy. If the enzyme or substrate rises in temperature it has been given more heat energy. This results in an increase in the substances kinetic energy so that it begins to move about more and more quickly. Both the enzyme and substrate will be in a liquid solution meaning that they can move about relatively freely. The more these molecules move about the greater the chance that the substrate will bump into the enzyme’s active site and thus the greater the chance that the enzyme will catalyse the break down of the substrate. This means that an increase in temperature will result in an increase in the rate of reaction. However, if the temperature increases a lot, the enzyme may become damaged. This is because the enzyme is moving about so quickly that some of the bonds (particularly the ones between the hydrogen atoms of the amino acids) begin to break. This can alter the shape of the enzyme and more importantly its active site, meaning that the substrate can no longer bind to it and be catalysed. Thus the rate of reaction rapidly decreases. In our experiment, it will be very difficult to control the temperature of the substrate and enzyme. However, if the experiment is carried out in similar conditions, the temperatures are unlikely to change significantly. By using a thermometer, we can measure the temperature throughout and thus take this into account when drawing conclusions. The temperature could be controlled by placing the apparatus in a water bath and adding hot of cold water accordingly, or using an electronically controlled water bath. This would be the fairest way to conduct the experiment. However this would be very difficult to do, as the apparatus is very large. Also as the temperatures are likely to stay very similar (as the experiments are carried out in similar conditions) the temperature can simply be measured and any fluctuations taken into account when drawing up conclusions.
- PH LEVEL- The pH of the conditions an enzyme is in can also affect its rate of reaction. pH is a measure of the acidity of a substance but is also a measure of concentration of hydrogen ions. Hydrogen ions can form bonds with the R groups of enzymes and can again change the shape of an enzyme’s active site. Enzymes are even more sensitive to pH than temperature because pH is a logarithmic scale of concentrations of hydrogen ions so a small increase in pH is a large increase in the amount of hydrogen ions present. This should not be a problem for our experiment. Like most enzymes, with the exception of pepsin which works in the stomach at a low pH of about 2, catalase works best in neutral conditions, which will be obtained by adding distilled water and not just water (which might be slightly alkaline) to the enzyme solution. pH can be easily measured and checked using litmus paper, which will not change colour in neutral conditions. The hydrogen peroxide may also affect the pH of the solution, so this must be measured when mixed with the celery solution.
- AGITATION- If the boiling tube containing the enzyme and substrate is shaken it will give the substances more kinetic energy. Like an increase in temperature, this will mean that the molecules are moving faster and the likelihood of the substrate colliding with the enzyme’s active site is increased. Therefore the amount the boiling tube is shaken must be kept constant throughout the experiment, or more accurately, the tube could be kept still throughout.
(Section on variables OCR Biology 1 textbook used as a reference.)
The dependant variable (rate of reaction) must also be measured. This can easily be done by collecting the oxygen produced by the breakdown of hydrogen peroxide when the enzyme and substrate are mixed. The oxygen can be collected in a calibrated tube full of water for a fixed period of time. The more oxygen collected in this time, the faster the rate of reaction. The rate of reaction can be calculated as follows:
Rate of reaction = 1 x volume of oxygen collected
Time oxygen is collected for
If the volume is measured in ml and the time in minutes, the unit for rate is ml per minute, which is written as ml min-1.
Preliminary experiment:
A preliminary experiment is needed so that the best concentration ranges of enzyme and the time to collect the oxygen for can be decided. Also the correct volume of substrate can be worked out so that it is in excess. The time that the oxygen is collected for needs to be large enough so that some oxygen is produced when the concentrations of enzyme are low. This time also needs to be large enough so as to reduce the percentage errors of the lowest concentrations of enzyme. If the volumes of oxygen collected are low, then any inaccuracy in our experiment is a large percentage of the total volume. For example an error of 0.5 ml, which would be small for a large volume of oxygen, is a percentage error of 10% if the total volume collected is 5ml. However, the time to collect the oxygen needs to be low enough so that the initial rate of reaction is measured. As the amount of substrate decreases, the rate of reactions for the different concentrations will change. To compare the rates of reaction for different concentrations it is fairer to compare the initial rate of reaction. Also the time needs to be low enough so that the oxygen produced can be collected in our collecting tube. Our concentration ranges need to be decided based on the same criteria.
A preliminary list of apparatus was drawn up based on our predictions and what we thought we needed:
APPARATUS:
Boiling tubes (X 10)
Delivery tube
Beaker (150ml) In the actual experiment a 250ml beaker will be needed.
Collecting tube (measuring up to 100ml)
Delivery tube (with bung)
Hydrogen peroxide 50ml In the actual experiment 150ml will be needed.
Distilled water 50ml In the actual experiment 100ml will be needed.
Celery extract 50ml In the actual experiment 100ml will be needed.
10ml syringes (X 3) In the actual experiment 2Х 5ml syringes and 1Х 10ml syringe are needed.
Thermometer
Litmus paper
Stopwatch
Clamp and stand
The apparatus was set up so that any oxygen given off by the substrate and the enzyme mixture in a boiling tube was delivered to the collecting tube full of water. The collecting tube (full of water) was placed in the beaker (also full of water) and clamped in place. The boiling tube was placed in a rack. We were given 5 M hydrogen peroxide. 10 ml of this was used in the preliminary experiment and this was enough not to limit the higher concentrations of enzyme. For the lowest concentration of enzyme, 0.5 ml of celery extract was measured out (using a syringe) and added to 4.5 ml of distilled water to give a 10% concentration. This was then poured into the boiling tube with the 10 ml of hydrogen peroxide. An intermediate concentration was used where 1ml of celery was added to 4 ml of distilled water to give a 20% concentration. The boiling tube was agitated the whole time throughout the experiment and the oxygen was collected for 30 seconds. After 30 seconds, the bung was taken off the boiling tube so that no more oxygen reached the collecting tube and the volume of oxygen was read off the tube and recorded. For the highest concentration, 5 ml of pure celery extract was added to the substrate, this was assumed to be a 100% concentration.
These results back up my prediction that as concentration increases, rate of reaction increases. However, shaking the boiling tube the whole time was difficult and unfair as sometimes the tube was shaken more. Also, the volumes of oxygen collected were small, meaning large percentage errors could occur. As the collecting tube only reads up to 0.1 ml, even for the largest volumes, this is a percentage error of 1% and for the lower concentrations, of nearly 15%. Because of this I decided to repeat the preliminary experiment so that I only gave the boiling tubes and initial shake (to get the reaction going) and increased the volume of celery solution to 10 ml. Therefore for the lowest concentration, 1ml of celery was added to 9ml of distilled water and for the highest concentrations, 10 ml of celery extract was used. The accuracy of this method of agitations was also investigated by repeating the experiment for the lowest concentration of enzyme.
These results show that this method is accurate and are better because the volumes collected are higher. The temperature remained constant (at about 18°C) and the pH was neutral, as the litmus paper did no change colour when it came into contact with the solutions, therefore these two factors should not affect the experiment. In the experiment large amounts of bubbles formed on the top of the boiling tube and some celery extract came close to passing through the delivery tube. The delivery tube will therefore need to be washed between experiments.
From the preliminary experiment, the concentrations and volumes of celery solution and substrate were worked out. Also the method and apparatus was refined. I decided that 5ml syringes would be better to use that 10 ml syringes when measuring out the lower volumes, because they are more accurate. Also, a larger beaker of 500ml is needed to place the collecting tube in so that it is totally submerged.
Method:
- The apparatus is set up as shown in the diagram above, so that the oxygen produced from the breakdown of the hydrogen peroxide was collected in a collecting tube full of water.
- Fill the collecting tube with water and place in a large beaker (250 ml) of water, and clamp into place.
- Measure out 10 ml of 5M hydrogen peroxide with a syringe. The 10 ml syringes measure to the nearest 0.5 ml, which is fairly accurate. Pour the substrate into a boiling tube, and place the tube into a rack. Keep this volume of substrate 10ml throughout the experiment.
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Add varying concentrations, but equal volumes of enzyme solution to the substrate. The lowest concentration is 1 ml of celery extract in 10 ml of solution (10% concentration) and the highest is 10 ml of celery extract with no distilled water added to it (100% concentration). Increase the volume of celery in solution by 1ml each time (and decrease the amount of distilled water accordingly). This results in an increases in concentration of 10% each time. Include a control experiment where there is no celery, but 10ml of distilled water, to see if the water has any affect on the substrate, which could affect the results. Measure out these volumes with a 5 ml syringe where possible, which is more accurate than a 10ml syringe because it measures to the nearest tenth of a ml. A table of volumes of celery and their respective concentration is shown below.
- Add the enzyme solution and immediately close the tube with the bung and start the stopwatch. Give the tube an initial shake. The oxygen produced should pass through the delivery tube and ‘bubble’ into the burette and not the beaker.
- After 30 seconds, the bung should be taken off the boiling tube and the volume of oxygen collected should be recorded. The initial rate of reaction for the experiments can be calculated by 1 divided by 0.5 minutes multiplied by the volume of oxygen collected.
Rate of reaction = 1 x volume of oxygen collected
Time oxygen is collected for
So for this reaction:
Rate of reaction = 1 x volume of oxygen collected (ml min-1)
0.5
- The delivery tube must be washed, the new water level on the burette recorded (so that the volume of oxygen for the next experiment can be calculated) and a new boiling tube used. The experiment is then repeated for all the other concentrations of enzyme.
- Time permitted, the experiments should be repeated so that an average can be taken and more accurate results obtained. Hopefully the results should be repeated at least 3 times so that 3 values for each concentration are obtained.
- The pH of the solution can be taken once and the temperature recorded so that these two factors can be taken into account when drawing conclusions.
Table to show concentration values for celery solution and their respective volumes of celery extract and distilled water.
In the interest of safety the experiment must be carried out in a careful manner. Hydrogen peroxide is a flammable substance but will not come into contact with any flames throughout the experiment. It is also a very powerful bleach and so should be kept away from the hair, eyes, skin and clothes. The enzyme may also be an irritant to the skin or eyes. If spillage occurs your hands must be washed immediately at the nearest sink and safety goggles must be worn. When using glassware, especially the brittle collecting tubes we must be careful.
When tabulating the results, volumes of celery extract and water as well as their respective percentage concentrations must be present along with the volumes of oxygen collected in a table. The temperatures, pH, total volumes of solution as well as the concentration and volume of hydrogen peroxide used must be noted. Averages of volumes of oxygen collected must be calculated and recorded. The rates of reaction for these averages must also be calculated and recorded on the table.
When plotting a graph of results, firstly amount of oxygen produced can be plotted against concentration, to give an overview of the course of each reaction. On this graph, the steepest part of the graph is the initial rate of reaction. Then rate of reaction can be plotted against concentration. For the graph, all 5 experiments should be plotted on the same graph so that comparisons can easily be made.
