pH – the pH will be kept constant because each enzyme works best at different pH levels. Most enzymes work best at a particular level of alkalinity or acidity. The pH alters the chemical properties of proteins, including enzymes. Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction
CONCENTRATION OF THE ENZYME – This will be changed. The more of the enzyme molecules produced by a cell, the faster the reaction will proceed (provided there will is enough substrate molecules available). Provided there is an excess substrate, an increase in enzyme concentration will lead to a corresponding increase in rate of reaction. Where the substrate is in short supply (i.e. it is limiting) an increase in enzyme concentration has no effect.
THE SUBSTRATE – This will be kept constant. An increase in substrate concentration will speed up the reaction if there are enough enzyme molecules to cope with the additional substrate.
In this experiment, it was easier to change the Concentration of the Enzyme, of the Substrate. I decided to change the Concentration of the Enzyme, because it was easier for me to add potato slices into the test tube than add more substrate. It was just quicker for me.
Method
My experiment will consist of 6 parts. I will take the reading of the amount of oxygen produced 6 times. Each time, the concentration of the enzyme will be different. It will be different because for each experiment, I will add one more slice on potato. I.E. I will take the reading of the amount of oxygen produced when there is 1, 2, 3, 4, 5, and 6 slices of potatoes. So for each full experiment, I will need a minimum of 21 slices of potatoes.
In order to conduct my experiment, I will do the following:
Cut the potato into small sizes of 0.5X0.5X1 (measurements in cm). I will cut them on the cutting tile, using the blade. I will then fill the trough full of water. I will then take the second measuring cylinder (with the higher readings), fill it with water, and then cover it with my thumb. I will submerge the measuring cylinder full of water under water in the trough, upside down. Once it’s in, I will take my thumb away. The measuring cylinder should still remain full of water.
I will then take a measuring cylinder, and fill it with 5cm3 of 20 Vols. Hydrogen Peroxide. I will be using this measurement of the substrate each time for each experiment. I will put the potato slice(s) into the test tube, and add the 5cm3 of 20 Vols. Hydrogen Peroxide into the same test tube. I will then take the Delivery tube and put the part with the bung into the test tube contain the enzyme and substrate. I will put the other end of it (with the opening) under water, under the measuring cylinder full of water. I will take the reading of the measuring cylinder before hand, and as soon as I put the other end of the delivery tube into position, I will start the stopwatch. I am going to see how much volume of gas is collected in 2 minutes. The amount of time I let the experiment go on for will remain the same, 2 minutes.
I will be conducting this experiment 3 times in total. This will give me a total of 18 readings (6 readings per experiment). I will do the experiment 3 times so that I can get a more accurate set of results. With just one experiment, I get one set of results. If I do it 3 times, I get a wider range of results, from which I can take an average. Also, it will minimize the ‘human error’ factor. No doubt that when I put the delivery tube under water, and start the stopwatch, there will be a time delay. This is called the human error. By conducting the experiment 3 times, the human error is reduced, and we can get a more accurate reading.
Safety
Great care should be taken whilst conducting this experiment.
Hydrogen peroxide can be harmful if swallowed. One should avoid drinking this, because it can cause vomiting, severe burns of the throat and stomach, and even death. Direct skin contact or breathing the vapors of hydrogen peroxide can also be harmful. So whilst conducting this experiment, gloves should be worn, and type of dust mask should be worn, so you do not drink it ( or get it anywhere near your mouth) and also so you do not inhale the vapours.
Laboratory coats should also be worn during the investigation to prevent chemicals from spoiling clothes. Care should also be taken whilst handling the chemicals as hydrogen peroxide is corrosive and the manometer fluid is permanently staining. Whilst using the razor blades, care should be taken to hold them by the handle and not the blade to prevent an accident occurring.
Planned Observations
I am planning to see how much of the oxygen gas is produced. The measuring cylinder which will be submerged under water will be full of water. I will take the reading of the cylinder. At the end of the experiment, I will take another reading. When I take this reading, I expect the amount of water in the cylinder to have dropped. This is because of the oxygen. The oxygen will bubble through the water, and ‘push the water’ out of the cylinder. From this, I can take a reading of the volume of oxygen produced.
I am expecting the amount of oxygen produced to increase as the amount of the enzyme increases. Similarly, if the amount of the enzyme decreases, then the amount of oxygen produced should decrease too.
I expect the graph of my results to look something similar to this:
Modifications to plan
Once I started conducting my experiment, I realized that I couldn’t cut the potato slices at the exact measurements each time. So I decided to cut one, and use it as a template.
I also noticed that the water in the cylinder that was submerged was never quite full. This meant that even though my thumb was covering the opening, the water was stick trickling out of the cylinder as I turned it upside down. This had an impact on my results. This made my results unreliable because when I took the final reading, there was already some empty space at the top of the cylinder. This added some volume to the final reading, which was not meant to happen. To combat this, I took the reading to start with. I took the reading for the second time, and this time, I subtracted the first reading from the second. This meant that I was now recording only the volume of oxygen produced nothing else
I decided to vary the enzyme concentration by varying the number of potato slices. However, although the enzyme, Catalase, occurs in the potato tissue, I did not know the exact quantity and certain slices might have more Catalase than others. This could be a major limitation in this investigation. I have tried to compensate for this, however, by taking multiple readings for each enzyme concentration so that inaccuracies are minimised once averaged.
Preliminary work was quite useful to me. It made me aware to the three mentioned problems, which could have affected the amount of oxygen produced. By doing preliminary work, it made me aware of the problems that I faced. This gave me a chance to get rid of the part which could have unnecessarily influenced my results. By doing preliminary work, my final readings were more accurate.
Observations
The data obtained from this investigation has been recorded in a table showing the Number of potato slices, Amount of oxygen produced and Observations. This means that the results of the experiment are presented in a clear and orderly fashion that allows patterns in the results to become more obvious.
These were the results obtained:
Experiment One:
Experiment 2:
Experiment 3:
The following table contains all 3 sets of readings, with the average amount of oxygen produced:
Limitations:
I decided to vary the enzyme concentration by varying the number of potato discs. However, although the enzyme, Catalase, occurs in the potato tissue, I did not know the exact quantity and certain slices might have more Catalase than others. This could be a major limitation in this investigation. I have tried to compensate for this, however, by taking multiple readings for each enzyme concentration so that inaccuracies are minimised once averaged.
Analysis
From the graph labeled ‘Graph to Show Average Amount of Oxygen Produced per Slice of Potato’, we can clearly see that as the number of potato slices increased, so did the volume of oxygen increase too. But, it only increased to a certain point. When there was 5 potato slices, the average volume of oxygen produced was 8cm3. Similarly, when there was 6 potato slices, the average volume of oxygen produced was 8cm3. This is due to something which is called a ‘limiting factor’. A limiting factor is something which limits the production of something. I.E. the Hydrogen Peroxide was a limiting factor in the production of oxygen. I can say this because we are increasing the number of potato slices, so this cant be the limiting factor. The hydrogen peroxide is limiting because there is too much of the Catalase enzyme, compared to the hydrogen peroxide. The amount of reactive hydrogen peroxide molecules is less than the amount of reactive enzymes. The limiting factor is shown on the graph at the top, when the curve levels out. This means that instead of the amount of oxygen produced increasing, the volume of oxygen produced is constant.
From the graph, I can conclude that the reaction was fastest when there was 5 or 6 potato slices. The experiment was quickest when there was more potato slices because there was the greatest number of free active sites available to the substrate molecules so that they could be broken down. The rate increased steadily from 0 to 5 potato slices and slowed beyond this point to give a "maximum level". It appears that at this "maximum level", increasing the enzyme concentration had little effect and other factors such as substrate concentration were limiting the reaction and prevented any further increases in the rate of reaction.
Conclusion - The results of this investigation are as I predicted in the hypothesis: " as the concentration of the enzyme increases (i.e. more of the enzyme) then the amount of oxygen produced will increase too ". The reasons for this are that there are number of variables that influence the decomposition of hydrogen peroxide in the presence of Catalase. Some of which can be classified as limiting factors i.e. the reaction is dependant or "limited" by their availability, to be able to function effectively; these include enzyme concentration, temperature and substrate concentration. All of these factors are required for an efficient reaction to take place, even when one is freely available the reaction can still be limited by the availability of the others. When I increased the enzyme concentration, it meant that there were more free active sites for the substrate molecules so that a greater quantity of substrate molecules could be broken down into products. However past a certain point, which in my investigation was at an enzyme concentration of 25 potato discs, there were many free active sites but insufficient substrate molecules to occupy them. Increasing the enzyme concentration further without increasing the substrate concentration has no effect on the rate of reaction which eventually will remain constant.
Evaluation
I think that under the circumstances, the experiment came up with fair results. With the apparatus I was working with, I think I obtained fairly accurate results, although the experiment could be made more accurate.
From the line of best fit from the second graph labeled ‘Graph With Line of Best Fit to Show Amount of Oxygen Produced’ it is clear that some of the points do not exactly fit on the line. They are anomalies. Although they have only slight inaccuracies (except the last two, which could possibly be big inaccuracies), they are an indicator of possible errors in the investigation. These may have occurred in the measurement of the quantities of the enzyme and substrate. Another possibility was that fluctuations in temperature caused the rate of reaction to increase. Although minimal contact was made with the apparatus during the investigation, slight undetected variations in the room temperature may have led to inaccuracies.
Possible Improvements/Alterations
To create a more accurate experiment in the future, several precautions or alterations can be made:
Instead of using potato slices that have slight variations in size, and volume of catalase, as a source for the enzyme, a 1 molar solution of the enzyme could have been diluted to create different concentrations. This way the concentrations can be measured far more accurately reducing the chances of errors in the investigation.
In a future investigation, a far greater number of enzyme concentrations between those already recorded should be tested reducing the extent of any anomalies on a graph where the line of best fit must be drawn.
In this investigation each reading was repeated so that an average rate of reaction for each enzyme concentration could be calculated. This could be improved by repeating the reading more frequently thus reducing the extent of any anomalies further, once averaged.
