Catalase
Hydrogen Peroxide → Water and Oxygen
Catalase
2H2O2 → 2H2O + O2
Catalase is able to speed up the decomposition of Hydrogen Peroxide because the shape of its active site matches the shape of the Hydrogen Peroxide molecule. This type of reaction where a molecule is broken down into smaller pieces is called a Catabolic Reaction.
- Preliminary Work
Before my main experiment, I conducted some Preliminary Work. This enabled me to fix the range and limits for the main experiment, and to decide on the different concentrations. I must note that 1 molecule of catalase breaks down 6,000,000 Hydrogen Peroxide molecules per minute. Therefore I will need very little yeast suspension
Apparatus:
- Conical Flask
- Beaker of 20 Volume Hydrogen Peroxide
- Rubber Bung with small tube, and one other hole
- Delivery Tube
- Large 500ml beaker of water
- Syringe
- Small Beaker of 20% Yeast Suspension
- Measuring Cylinder (plastic, calibrated)
- Constant Water Supply
In the Preliminary Work, I first timed approximately how long it took for the oxygen to stop being produced (i.e. the water to stop bubbling). Here are the results:
However, I stopped there, because I realised that rather than
waiting for the bubbles to stop, which was an unfair test due to shaking the flask, I should measure a particular amount of Oxygen, say 25 cm3 displaced in a measuring cylinder.
Here are my results:
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Full 20 Volume 40 cm3 Peroxide, with 2 cm3 of Yeast Suspension – This reaction was too quick to measure, the flask filling with reacting yeast which flowed into the delivery tube.
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40 cm3 of Peroxide, with 1 cm3 Yeast – This reaction was quite fast, taking approximately 20 seconds. This, I feel is an adequate starting point.
I then decided that 1 cm3 of Yeast each time would be sufficient.
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20 cm3 Peroxide with 20 cm3 Water, with 1 cm3 Yeast – Lasting more than 1 minute – again adequate.
-
25 cm3 Peroxide with 15 cm3 Water, with 1 cm3 Yeast – Longer still, but a reasonable time.
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5 cm3Peroxide with 35 cm3 Water, with 1 cm3 Yeast – Too long, longer than 7 minutes.
Therefore, I concluded that we should time the time taken to collect 25 cm3 of Oxygen, with a constant 1 cm3 Yeast, and a substrate volume of 40 cm3, varying the cm3 ratio of Peroxide : Water, from 40:0, to 10:30. I also decided that rather than holding the measuring cylinder, I would use a clamp/stand. Lastly, to measure the numbers more carefully, I decided rather than calculating the concentration in ratio of water: peroxide form, I should use percentages. This will be much more accurate.
- Prediction
I predict that as substrate concentration increases, the rate of catalysis will also go up, at a directly proportional rate until, as my research shows, the solution becomes saturated with the substrate hydrogen peroxide. When this saturation point is reached, then adding extra substrate will make no difference, and the rate will remain constant.
At a constant enzyme concentration and at lower Peroxide concentrations, the substrate is the limiting factor. As the substrate concentration increases, because more active sites of the enzyme are being used up, rate steadily increases, due to more reactions and consequently more oxygen. Once, however, the number of substrate molecules added exceeds the number of active sites available, the rate will no longer go up. This is because the maximum numbers of reactions are being performed at once so any extra substrate molecules will have to wait until some of the active sites become free.
- Method
Apparatus for Main Experiment:
- Conical Flask
- Beaker of 20 Volume Hydrogen Peroxide
- Rubber Bung with small tube, & one other hole
- Delivery Tube
- Large 500ml beaker of water
- Syringe
- Small Beaker of 20% Yeast Suspension
- Measuring Cylinder (plastic, calibrated)
- Constant Water Supply
- Clamp and Metal Stand
- Pipette
To test how the concentration of the substrate, Hydrogen Peroxide, affects the rate of reaction, I set up the apparatus as shown below.
-
Add 1cm3 of Yeast into the syringe. Add 40 cm3 of Hydrogen Peroxide solution at a concentration of 20% into the conical flask. Use a pipette to measure out the volumes of the yeast as accuracy is important to make it a fair test.
- Attach the syringe to the rubber bung. Fix a delivery tube to the other hole. Place the end of the delivery tube in a beaker of approximately 400ml of water.
- Using a piece of tissue, over turn a measuring cylinder full of water into the beaker of water. Carefully position it
directly above the tube, ensuring that the open end of cylinder does not break above the surface of the water.
- Firmly attach the bung onto the conical flask. Inject the yeast suspension from the syringe into the flask. At the same time, start the stopwatch.
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Bubbles should start rising from the tube, and the measuring cylinder’s water should begin to get displaced. As soon as 25cm3 of water has been displaced (note the meniscus), stop the stopwatch, and note the elapsed time to the nearest 1/10 of a second.
- Repeat the experiment with hydrogen peroxide concentration of 16%, 12%, 10%, 8%, 4% and 0%. The 0% concentration of hydrogen peroxide solution is done as a control solution to show that at 0% concentration no reaction occurs. The different concentrations are made by adding tap water to the 20% Hydrogen Peroxide in the correct amounts, shown below.
- I shall repeat all the tests at least three times so that an average could be obtained. Repeating the experiments several times help to produce better and more accurate
results, and any inaccuracies in one experiment, will be compensated for by other experiments. I shall record the results in a table such as the one below:
I have decided to calculate the rate too, as this would enable me to draw a conclusive graph. The rate can be calculated by:
Rate = 25/Average Time (s)
This will give the rate of Oxygen produced per second, because I calculated the time taken to collect 25cm3 of Oxygen.
I will make sure the conical flask is thoroughly washed out and dried so that there will be an exact amount of water/peroxide. If I do not do this, it will be an inaccurate investigation.
I have to also note that for safety, the maximum concentration of Hydrogen Peroxide available to me is 20%. Safety is paramount in this experiment, as higher concentrations of Peroxide could harm the skin. However, even at this concentration, Peroxide will be corrosive to benches, and more importantly eyes. If it makes contact with the benches or my skin, I must cleanse it off immediately. To prevent it from entering my eyes, I shall wear safety specs or goggles.
- After I performed the first experiment, I realised a fault in my method. When I inserted the yeast via the syringe, I found that not all the yeast was transferred into the conical flask. Therefore, after I sucked up the yeast into the syringe, I removed the end from the beaker of yeast, and carried on pulling on the end, so that the plastic black end met the ’10 ml’ mark. This left me with air behind the yeast. Therefore, when I inserted the yeast, the extra air helped to push ALL the yeast in. I started again.
Results
All the times are in seconds. The average results are all written down to one decimal place because although the stopwatch gives results to two decimal places it is impossible to get accurate times to two decimal places due to the fact that our reaction times are not fast enough to stop the stopwatch precisely. I then worked out the rates of the reactions with the equation
Rate = 25/Average Time
The number 25 is used because we collected 25cc of gas. This divided by the average time will give us the rate at which hydrogen was produced per second.
I have highlighted the two sets of anomalous results, in red. These, I only found were anomalous, after plotting the graph.
Also, I found that one results from the 12% concentration column was anomalous. I noticed this immediately. Therefore I repeated that part of the experiment. I have noted the second and useful result, as normal, in green. Also, I have noted the anomalous result in red.
Analysis
2.
My experiment shows me that as concentration increases, rate of oxygen increases, with direct proportionality, until approximately 16 % concentration of Peroxide.
When the concentration is doubled from 8% to 16%, the rate goes up from 0.48 to 1.97. This is an increase of 1.49. Therefore, I would expect the rate to increase two fold again if the Peroxide concentration is increased two times, because there are twice as many substrate molecules which can join onto the enzyme active sites.
3.
The reason that the rate is less than two times could be due to the fact that at 16%, the enzyme’s active sites are, or becoming close to, saturated with the Hydrogen Peroxide. Another way of reasoning this is simply because of experimental error which causes inaccuracies.
After 16%, you can see that the gradient of my graph starts to flatten out. This is because the active sites are saturated. Increasing the concentration will not increase the rate because all the active sites of the enzyme are being used, Therefore, the substrate molecules have to wait there turn until an active site is free. At this point, the only way of increasing the rate would be to increase the availability and hence the volume of Catalase.
If my first theory stated above applies, I can say that my results supported my prediction, and the reaction performed as explained theoretically in my plan.
The Lock and Key theory is shown successfully here. It is clear that as more molecules are present, more active sites of the enzyme are filled, and therefore, more Hydrogen Peroxide broken down into Hydrogen and Oxygen.
Also, my experiment shows that as stated in the plan, any one enzyme will only break down one substance. Therefore, standing by this, I was able to add water to alter the concentration, with the knowledge that it did not affect the results.
4.
However, I can note from my graph, that the rate never truly level off – it is still a gradual curve, showing an increase in rate. Therefore, if I were to change my variable, concentration of Hydrogen Peroxide, over a much larger range, say up to 45%, I would expect to see that the rate would no longer increase at all, but would be a flat line, due to my theory above.
Lastly, my results also support my prediction and theory, because of my result at 0% concentration of Peroxide. I said that because there would be no peroxide molecules to attach onto the active sites of the enzymes, at 0% concentration, there would be no reaction. This, again, is because catalase in yeast can only break down peroxide, so water molecules can not fit into the specific shape of the active site. Therefore, there was no oxygen produced, although there is oxygen in water.
The last thing that I can point out in my analysis is that although the theoretical maximum rate of reaction is when all the sites are being used, in reality this theoretical maximum is never reached due to the fact that not all the active sites are being used all the time. The substrate molecules need time to
join onto the enzyme and to leave it so the maximum rate achieved is always slightly below the theoretical maximum. Therefore, I can say that the time taken to fit into and leave the active site is the limiting factor in the rate of reaction.
I must take account of this, for my evaluation.
Evaluation
1.
I feel that my experiment was a definite success. I think I have solved the investigation well, and I think that my conclusion in my analysis is justified, because my results so closely fit my prediction and scientific research on the activity of an enzyme.
Also, I think my results are quite reliable. I say that due to the fact that all the points, except the last fit quite closely to the line of best fit. The last does not, because of the saturation that I mentioned in my analysis. However, I would take more results around the 8% - 10% area because these two dip slightly below the line of best fit.
However, I am not wholly confident with the information the graph has given me, because as I said in the analysis, only if I performed the investigation with a larger range, would I be able to say whether my graph really would level off because of active site saturation.
2.
One other concern is my accuracy in the experiment. I did whatever I could to ensure accuracy, but there are a few limitations that could not be avoided
To help make this experiment more accurate, I repeated it three times and then used the average of all the results to plot a graph with a line of best fit. I tried to keep all the variables except for the concentration of Hydrogen Peroxide the same for all the experiments. Here are a few:
a) There is a slight delay between pouring the yeast into the Hydrogen Peroxide, and starting the stopwatch, and then seeing the oxygen bubbles rise from the water into the tube. This will slightly affect all the results but as I carried out all the three steps in the same way for all the experiments it should not make any difference to the overall result.
b) It is also impossible to precisely measure out the amounts of Hydrogen Peroxide, Yeast and Water each time. As the scale on the pipettes shows the volume to the nearest mm3 the volume of the solutions that I used should be correct to the nearest mm3.
c) Due to the fairly slow speed of our (human) reactions it is only possible to measure the time of the reaction to the nearest 0.1 second even though the stopwatch shows the measurements to the nearest 0.01 second. Regarding this, digital meters also have an error or +/- 2%. This, added to my slow human reactions, make my results inaccurate.
d) Because I was collecting the gas in a test tube, I had to make a decision when to stop the stopwatch. Water has a meniscus, so when reading off the water level, my judgement in different experiments of the investigation could have been slightly different- another source of unavoidable error, because the water level sometimes went down very quickly, so very little time was available to make a decision.
3.
Now I shall discuss my anomalous results. I have highlighted all my anomalous results in red on my results table. As I have mentioned above, the sets of data at 8% and 10% are slightly below the line of best fit, on my graph. They do not change the conclusion of my results, and are only slightly irregular, so I think that they are due to experimental error on my behalf.
Apart from these, I have only one other anomalous results: Test 3 in the 12% experiment. This was quite far off from the other two, so I repeated the concentration again, and took the average excluding the anomaly.
4.
To improve this experiment, I would do several things.
a) I would have another person, as a helper. This, I think, would eliminate the problem of inaccuracies regarding the starting of the stopwatch.
b) Rather than collecting the gas in a test tube of water, I would collect it in a gas syringe. This would cancel a need for a clamp and stand, and also make reading off the amount of gas collected easier, getting rid of some inaccuracies.
c) Rather than use yeast as a source of catalase, I would use catalase itself in its pure form. This could be a more accurate way of obtaining conclusions and untainted results.
d) The experiment could be repeated more times to help get rid of any anomalies. A better overall result would be obtained by repeating the experiment more times because any errors in one experiment should be compensated for by the other experiments.
e) Using more concentrations of Hydrogen Peroxide would have produced a better looking graph, which would be more accurate.
5.
As extensive work, I would like to use concentrations higher than 20% to extend the graph so that the maximum possible rate of reaction could be reached.
Also, to let me get a better picture of how substrate breakdown by an enzyme is affected by substrate concentration, I would repeat the same experiment, with a different enzyme, and therefore, a different substrate. I would be able to see if the same conclusions can be made.
