Distilled water - to wash the beads in
Strainer - to separate the beads in
Hydrogen Peroxide (20vol) 20cm per test - substrate
Stop clock 1 sec - to time 30seconds each test
Measuring cylinder 1cm 25cm - to measure oxygen released
Water - to be displaced
Clamp stand - to hold measuring cylinder in place
Delivery tube and bung - to deliver oxygen
Measuring cylinders, 4 1cm 50cm - to measure Hydrogen Peroxide, Yeast solution, Sodium alginate solution and calcium carbonate in
Conical flask 2.5cm 50cm - for the reaction to take place in
Beaker 50cm - for mixing the sodium alginate and yeast solution in
Beaker 100cm - to drop beads in to
Stirring rod - to mix the yeast and sodium alginate solution together
Tub - to contain the water to be displaced
Method
20cm of yeast solution and 20cm of sodium alginate solution are to be measured in the 50cm measuring cylinders and then combined in the 100cm beaker and mixed thoroughly with the stirring rod. This is then to be drawn up into a syringe and dropped at a regular pace into 50cm of calcium chloride in a 100cm beaker. After being left for 3-5 minutes, when set, the beads are to be strained off and washed thoroughly with distilled water. A 50cm conical flask is to be filled with 20cm Hydrogen Peroxide, measured in a 50cm measuring cylinder, and 10, 20, 30, 40 or 50 immobilized Catalase beads added. A bung with delivery tube is to be attached to the 50cm conical flask as soon as possible and the delivery tube is to be put in the upturned and water filled 25cm measuring cylinder, held by the clamp stand. As soon as the bung is attached the stop clock is to be started and 30 seconds is to be measured. When this time is reached the results are to be observed and recorded. 3 repeats of each value of beads are to be performed and a mean value found, to hopefully stem any errors. Also a control experiment is to be done with 50 beads which were boiled to denature the enzyme, thus proving that it is the enzyme that has the effect on oxygen production and not any other factor.
It is essential that the following variables are controlled and kept constant during the entire experiment:
~ 30 seconds for each experiment - by timing with the stop clock (accurate to 1 second)
~ The same person must read the results and carry out each stage of the experiment - one person does the experiment, therefore controlling this factor
~ Accurate number of beads - these are counted and then re-counted
~ The temperature of the substrate the temperature of the enzyme - they will be constantly kept at room temperature
~ The pH of the substrate and enzyme - the pH will not change throughout the experiment
~ Time taken between the stop clock being started and the addition of the Hydrogen Peroxide to the yeast beads, and connection of the delivery tube and bung - this will be difficult to control but every effort will be made to start them simultaneously.
These variables are to be kept the same because they will affect the outcome of the experiment and may affect how much oxygen is released.
The results are to be recorded in a table and a graph showing the mean value of oxygen released for each number of beads.
Risk Assessment
The enzyme is of a very low risk, but the Hydrogen Peroxide is corrosive and an irritant so eye protection must be worn at all times, as well as a lab coat. If there was to be a spillage of Hydrogen Peroxide, it is essential that it is cleared away immediately, it should be mopped up with a dry disposable towel and then the surface washed thoroughly with water. If any solution spilt onto skin or into the eyes, then the area should be washed immediately with plenty of water. If irritation persists then medical help should be found. If any is swallowed then medical help should be found immediately.
There are no ethical implications in this experiment.
Results
Number of yeast beads Amount of Oxygen collected - Repeats (cm ) Mean amount of oxygen collected (cm )
1 2 3
10 3.5 3.5 3.4 3.5
20 6 7 6 6.3
30 7.5 6.5 7 7
40 9.5 10.5 10 10
50 14 15.5 12 13.8
control - 50 0 0 0 0
Conclusion
The graph shows that as the number of beads rises, the oxygen released also rises. This shows that the more beads there are the quicker oxygen is released. The Catalase present in the yeast beads is the only enzyme that breaks down Hydrogen Peroxide. The results show that the more Catalase active sites, then the quicker the Hydrogen Peroxide substrate is broken down into oxygen. Enzymes are very complex 3 dimensional globular proteins. The small part of the enzyme that comes into contact with the substrate (in this case Hydrogen Peroxide), is called the active site, it is theorized that enzymes work with a "lock and key" mechanism (the enzyme's active site being the lock, the substrate being the key). The active sites on the Catalase enzyme are filled with the Hydrogen Peroxide, the more enzyme there is then the more active sites there are, and so therefore the more Hydrogen Peroxide can be broken down in into oxygen in a certain amount of time. The active site of an enzyme can be used again and again; therefore enzymes work best in low concentrations, providing that the temperature, pH and other variables are at their optimum for that enzyme. The graph may show an anomalous point, as marked, which could mean inaccuracies in procedure or results, this is commented on in the evaluation. The range bars also on the graph show some variation between results. The graph plots an average which should combat any extreme variation.
As I theorized in my hypothesis, the more beads present, the more oxygen is released as a product of the breakdown of Hydrogen Peroxide.
Evaluation
The repeats are quite consistent at the beginning of the experiment, but in the 50 beads experiment, the range of values attained in the repeats seem to be quite separate from one another, thus shown on the graph by the distance between points on the range bar. This could mean an inaccuracy in the experiment and could cast doubt over the reliability of the results. There is also an apparent anomalous point, as indicated on the graph, on the 20 beads experiment, this could also mean inaccuracies. The range bars displayed on the graph, show that there is variation between some results in the tests. This obviously means inaccuracies that need to be dealt with in further experiments.
The measuring apparatus used could have been more accurate, this of course causes inaccuracies within results and causes inaccurate implementation.
The temperature, as known to affect the rate of reaction of enzymes drastically, was always kept at room temperature, though, as other experiments were taking place at the time, it is likely that heat released from their experiments may have made the room temperature fluctuate slightly, this in turn would affect the enzyme and substrate temperature. This change would have only been slight, and as the entire experiment would have been carried out under these conditions, it is unlikely that it had any affect on the final outcome.
The pH was kept constant throughout the experiment, as no other substances came into contact with either substrate or enzyme. The immobilized enzyme was thoroughly washed with distilled water, of neutral pH, to remove any calcium carbonate.
This experiment was difficult to control as when adding the Hydrogen Peroxide to the beads the delivery tube and bung may not have been attached simultaneously and the stop clock may not have been started simultaneously. This means that the oxygen produced at the beginning may have been lost as the delivery tube was not attached simultaneously, and so would affect the results of the experiment, as less would be measured than produced. This effect would be the same in each experiment, so to compare results to one another would be fair. It would be easier to re-do the experiment with a helper to push the "start" button on the stop clock, whilst the experimenter added the Hydrogen Peroxide to the beads and attached the delivery tube and bung to the conical flask.
The gas collected at the beginning would be air displaced from the delivery tube and conical flask and not oxygen, this however is considered acceptable, as the air displaced would be equal to the oxygen produced, though this point must be noted, as the experiment I studied was that of oxygen released, and not air displaced and measured in its place.
Whilst all attempts were made to make a constant regular shape and size of beads, it is probable that there were different sizes of beads. If the beads were smaller then less immobilized enzyme would be present per bead, and so therefore affecting the amount of active sites present and reaction rate attained. If the beads were bigger then the active sites would be increased and so the reaction rate would increase too. This experiment is but one and you can not say that the results are conclusive because different samples would produce different results each time. It is also just studying one enzyme, and so the results are not generalisable to other enzymes, though it is probable that the same general trend is shown in all enzymes. As such a small set of values were used, the experiment does not fully answer the question of whether the volume of enzyme affects the rate of reaction.
The experiment performed had to be of a smaller scale, to avoid spillages and risks of over flowing substrate. In a larger scale experiment, wider values could be explored to hopefully extend upon the conclusion and find additional evidence.
