Food Processor and Scalpel: It is used to cut the potatoes into 1cm discs. This will make it easier to get all the content that could possibly be obtained from the potato.
Inverted Measuring Cylinder: I will use it to measure the rate of release of the oxygen gas by seeing how much oxygen gas was forced to the end of the cylinder, by measuring the volume; I will be used to determine a relatively accurate measure of the amount of oxygen released in the decomposition of the substrate hydrogen peroxide.
Measuring cylinders have a 2.5% error range; this is not enough to affect the investigation.
Petri-Dishes: I will use the Petri-dishes and distilled water to hold already cut potato discs, this will prevent dehydration of the discs.
Variables
Apart from enzyme concentration, the variables that could affect the enzyme activity of catalase are controlled so that they do not affect the experiment. These variables are:
- Temperature
- pH
- Substrate concentration
- Presence of enzyme inhibitors
Temperature
As temperature increases, rate of reaction increases. To control this variable, I will maintain the set-up at room temperature. Also I will make sure I handle the test-tubes with a tong and put it in a test-tube rack when it’s not in use to prevent any heat from my hands from affecting the catalase. I chose to use room temperature for convenience and although room temperature might not be constant, the experiment was performed in a short period so there is little or no change in the atmospheric temperature.
pH
Enzymes react to changes in pH. Catalase has ph7 as it’s optimum pH. To control this variable, I will keep the pH of the experiment constant using a pH7 buffer. I chose to use the pH7 buffer because it maintains a suitable pH level for catalase to work properly. Also it simulates the pH of the natural environment (potato) of catalase.
Substrate Concentration
An increase on substrate concentration results in an increase in the rate of the reaction.
To control the substrate concentration, I will use 10cm3 of 30vol hydrogen peroxide all through the investigation.
Surface Area for Enzyme Activity
The area with which the enzymes react is also an important factor in enzyme reactions. The higher the area exposed to the substrate, the higher the rate of reaction. I controlled this factor by making use of a cork borer and cutting the potatoes into equal disc thickness. By so doing, the area of potatoes exposed to the substrate in each disc is equal.
Enzyme Concentration
The factor I am going to examine is the effect of enzyme concentration on the activity of the enzyme catalase. As the enzyme concentration increases, rate of reaction increases.
How I Plan To Take My Readings
I plan to vary the enzyme concentration and see how it affects the rate of catalase activity. Since catalase is found in potatoes, I plan to use the amount of surface area exposed to the substrate to vary the enzyme concentration.
I will use a cork-borer to take out cylinders of the potatoes; this is to ensure all the discs have the same diameter. I will cut the cylinders into 1cm discs to make all the discs of equal total surface area.
Total Surface Area of Disc = 2Πr2 + Πdh
T.S.A= [2 X Π X (½ X Diameter of Cork borer)2] + [Π X (Diameter of cork-borer) X Thickness of disc]
Π=3.2
Diameter of Cork-Borer= 12.5mm = 1.25cm
Thickness of Disc= 1cm
T.S.A of one disc= [2 X 3.2 X (½ X 1.25)2] + (3.2 X 1.25 X 1)
= 2.5cm2 + 4cm2
= 6.5 cm2
Therefore, one disc of potato exposes 6.5cm2 of catalase to the hydrogen peroxide. Five discs of potato expose 32.5cm2 of catalase to the hydrogen peroxide. The higher the surface area of exposed potato, the higher the concentration of potato. By varying the discs in fives, I will be able to investigate the rate at which the volume of oxygen released changes with the steady increase of enzyme concentration.
The higher the catalase enzyme concentration, the higher the volume of gas released into the inverted measuring cylinder.
The rate of reaction will be measured by measuring the amount of water displaced by the oxygen in one minute. By dividing it, the amount of oxygen released per minute gives me the rate of enzyme activity.
I will report my readings in a reading in this form.
Risk Assessment
- An overall should be worn, because the hydrogen peroxide is a dangerous chemical and so gloves and goggles will have to be warn too, at all times, so that it does not react with skin or get into our eyes. If it does by any changes, eyes must be washed immediately with warm water and the teacher should be informed.
- Hair should be tied back so that it does not come in the way as a distraction.
- The apparatus should not be kept too close to the edge of the bench, in case anything spills.
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The side arm test tube should be supported; maybe with a retort stand or test tube rack, the other test tube should be placed in the same test tube rack and kept away from the edge of the bench.
Hydrogen peroxide is a very dangerous solution; it can burn through clothes, bench surfaces and even skin, and therefore all safety issues should be taken into account and not ignored.
Environmental and Ethical Issues
Potatoes will be used; as they do not contain blood (as liver) therefore there is no problem of infection.
Moreover, there is no problem with animal rights or religious concerns. Muslims might object to pork and Hindus to beef.
METHOD
I used a cork-borer to take out samples of potatoes from the tubers. After putting the samples on a tile, I used slicer to hold the samples while I cut the potatoes into equal disc lengths. I placed the discs under distilled water in the seven different petri- dishes to prevent dehydration or contamination of the discs. Each petri-dish contained a different number of potato discs (5, 10, 15, 20, 25, 30, and 35).
I filled a beaker to the brim with water, I then filled a measuring cylinder with water and while carefully blocking the tip, I inverted it into the beaker. I placed one end of the delivery tube into the open end of the inverted measuring cylinder.
I started up by using a syringe to measure 10cm3 of hydrogen peroxide into a test–tube; I used a different syringe to measure 10cm3 of ph7 buffer solution and added it to the test-tube contents.
After completing all that, I emptied the potato discs into the test tube and covered it with the bung of the delivery tube and waited for five seconds. I did this to prevent measuring the gas released due to the air displaced initially by the pressure forced in when I pushed in the bung.
After the initial rush, I started the timer and closely watched the amount of air evolved by watching the amount of water displaced from the inverted cylinder. Immediately the time reached 60 seconds, I removed the dispenser from the measuring cylinder and checked the amount of gas released by seeing the level of water displaced.
Experiment Apparatus
Precautions
- The initial rush of oxygen caused by a change in pressure as the hydrogen peroxide is added to the flask is ignored and I started the timer once this initial rush had passed.
- I constantly agitated to contents of the test-tube to ensure the catalase is evenly distributed throughout the solution, so that the enzyme-substrate complex is formed.
- The airtight bung was used to ensure other atmospheric molecules do not mix with the contents of the tube.
- I will use a fresh unused test-tube for each set of potatoes to ensure that there is no cross-contamination of the solutions.
- I placed the already cut discs under distilled water in the petri-dishes to prevent dehydration or contamination of the discs.
- I wore gloves while performing the experiment to make sure I didn’t contaminate the potato discs.
RESULTS
Table of Results Comparing Number of Potato Discs To Rate Of Reaction
Table of Results Comparing Surface Area To Rate Of Reaction Of Catalase
Trends And Patterns
The first graph shows the activity of catalase with different amounts of potato discs to show how the volume of oxygen produced varies with different enzyme concentration. In this graph, there is a steady increase in the volume of oxygen released as the concentration increases.
The second graph shows the effect of varying surface area of catalase on the rate of reaction, as expected; the rate of reaction increases as surface area increased which proves the fact that the increase in enzyme concentration is dependent on the amount of surface area of the potato exposed to the substrate.
Explanation
Referring to the graphs, all things being equal, the rate of reaction should be directly proportional to the number of potato discs used. This suggests that if the enzyme concentration were increased, the rate would also increase. It is due to the fact that as there are more hydrogen peroxide molecules around, the more often the enzyme’s active site (catalase) can bind with one, thus releasing products of water and oxygen gas.
Again referring to the graphs, it shows a steady increase in the rate, almost proportional to that of the increase of enzyme concentration. The rate of reaction starts off very low at the first stages. This is because there are fewer enzymes within a certain area of the solution and so less of the particles, when given the necessary amount of kinetic energy (by agitating the mixture), move close together. So, fewer collisions occur between particles, in this case, the enzyme catalase and the substrate hydrogen peroxide; therefore fewer reactions occur at this particular point.
By increasing the enzyme concentration(number if potatoes), the amount of particles within a certain area in solution also increases; therefore, more of the particles would collide with one another due to the fact that they are closer to one another, thus more reactions between catalase and hydrogen peroxide occurs, resulting in an increase of the rate.
In conclusion, it is safe to state that by increasing the enzyme concentration it would result in an increase in the rate of reaction; the rate at which the enzyme breaks down hydrogen peroxide to water and oxygen gas.
Discussion and Evaluation
The results of this experiment were relatively consistent. This can be due to the fact that careful attention was paid to details and also following all precautions to make sure the results were accurate and reliable.
Despite the accuracy, I noticed that the amount of oxygen released at 5 potato discs was relatively distant from the rest of the curve. This can be explained with the fact that there was a higher percentage error that is produced when the gas released from the mixture escapes before the plunger is fitted in.
Although this did not affect the rest of the reading, it affected the first reading more because the amount of oxygen lost is high when compared to the amount actually released by reaction.
Also, there might have been temperature variations during the experiment as I did not carry out the whole investigation in a water bath. My decision not to use the water bath was because it might have caused errors because the effect of temperature might not evenly spread across each test tube. To make sure temperature does not affect the reaction, I used gloves throughout and made sure I did the experiment in a short period of time to make sure there was little or no change in the temperature.
In all, I think for a minor investigation my experiment went as expected but this cannot be fully ascertained as it is not an in-depth view of the factors.
Criticisms & Ways to Improve Experiment
Instead of using potato discs that have a slight variation in size and volume of catalase, as an enzyme, I believe it would have been easier and less error-prone to use a 1molar solution of the enzyme catalase for the experiment. This will reduce chances of errors in the investigation.
In this experiment, I only investigated five enzyme concentrations. Although there was a large range, there were insufficient measurements between the readings creating a gap where guess work was what I used to predict the rate of reaction at these points. If I were to do this experiment again, I would measure more rates of reactions between the readings already measured.
In future investigations, a far greater number of enzyme concentration between those already recorded will be taken into account. Because in this experiment only five enzyme concentrations was considered thereby creating a gap where the rate of reaction at these points needed to be assumed. But having a larger range and a greater number of enzyme concentration values, the graph should would be clearer with more trends, this will improve the accuracy of the experiment.
To broaden the scope of the investigation, and to add more information to the enquiry, I could test the effect of enzyme concentration on the rate of this same reaction- decomposition of hydrogen peroxide by catalase. But it time, I would test the time taken for the reaction to be completed instead of how much of the oxygen was released in one minute.
