Enzymes such as Catalase are large protein molecules that are found in living cells. They are used to speed up specific reactions in the cells. They are all specific as each enzyme just performs one particular reaction. In their globular structure, one or more polypeptide chains twist and fold, bringing together a small number of amino acids to form the active site, or the location on the enzyme where the substrate binds and the reaction takes place. Enzyme and substrate fail to join together if their shapes do not match exactly. This ensures that the enzyme does not participate in the wrong reaction. The enzyme itself is unaffected by the reaction. When the products have been released, the enzyme is ready to bind with a new substrate. Enzymes work to change the rate of a reaction without being absorbed by the reaction. The reactant that an enzyme acts on is called the enzyme’s substrate. The product is what the enzymes convert the substrate into.
Catalase is an enzyme found in food such as potato and liver. It is used for removing Hydrogen Peroxide from the cells. Hydrogen Peroxide is the poisonous by-product of metabolism. Catalase speeds up the decomposition of Hydrogen Peroxide (which is the substrate) into water and oxygen as shown in the equations below:
Hydrogen peroxide + Catalase Water + Oxygen
H2O2 + Catalase H2O + O2
It 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 an anabolic reaction.
Potato split into 4g tubes (enzyme catalase)
A pilot test was carried out to figure out how much and in what form the apparatus would need to be in. This is what was concluded; the potato should be ground down with sand (which is unreactive with H2O2) into a paste. This gives a larger surface area that permits the reaction to go faster because the substrate molecules can get to the enzyme molecules faster and more efficiently.
The reaction will be timed as soon as the solution (H2O2 and H2O) is added. The oxygen that evolves will be measured and recorded every 30 seconds until the reaction finishes.
The concentration of the solution will be variegated in the experiment. This is the only element that is varied everything else must stay the same to keep the investigation unbiased. 25cm3 of solution was chosen to be used because in the pilot experiment this was all that was necessary to produce a sufficient amount of oxygen in the gas syringe so that it could be easily measured. If there were more solution it would take longer to pour in to the conical flask therefore gas would escape before the rubber bung could be fixed into place to seal the conical flask. If less solution were to be used the reaction would take longer because there wouldn’t be as many substrate molecules to react with the enzymes.
The solution will be measured in a measuring cylinder. The range was selected because it is easily multiplied into 100, which is what is needed to calculate percentages. The measuring cylinder was chosen to be used over all other methods of measuring because it is easily available.
Percentage of H2O2Volume H2O2 (cm3)Volume of H2O(cm3)
The enzyme catalase works best at a warm temperature so to speed the experiment up the conical flask will be in a water bath at the temperature range 40-45 oC. The water bath needs to be kept at a constant temperature because the enzymes will then be working at the same constant rate. The mass of the potato needs to be kept the same at 4g so there is the same amount of enzyme in each experiment. The overall amount of solution needs to be kept constant to ensure that the % concentration is correct.
The oxygen evolved will be measured every 30 seconds from the moment the solution is added to the potato. This will be done so that the results from each experiment can be compared at the end.
The experiment will need to be repeated at least 3 times to allow for an average to be taken, and to be able to exclude any anomalous results and still obtain an average. Overall the experiment will be done 18 times. Although it will be permitted to carry the experiment out a few more times if one proceeds to go wrong.
The results will be recorded in a table that is clear and easy to understand.
Here is a sample of the table that will be used:
Time (s)Experiment 1Experiment 2Experiment 3AverageRate of reaction
The results will be plotted into an evolved graph and a rate of reaction graph. Rate of reaction is measured by Average
Once the graphs have been drawn trends will be looked for and identified.
Once the Hydrogen peroxide is added to the enzyme catalase (potato) it should begin to fizz violently. This is a sign that the two substances are reacting together. This will show that the catalase is decomposing the hydrogen peroxide so fast that it can be seen. The enzyme catalase and the substrate hydrogen peroxide will join together to make the products oxygen and water.
This is called the lock and key method. The enzyme catalase is specific to this reaction.
Because the reaction is being heated in the water bath it will go much faster and be easier to see the increase in oxygen evolved. It will also allow for a third replicate to be taken because the time allocated for the investigation is brief and without the reaction being speeded up there may only be time for one or two experiments for each concentration.
As the substrate concentration increases, the rate should go up at a directly proportional rate until the solution becomes saturated with the substrate hydrogen peroxide. Once the saturation point is reached, adding more substrate will make no difference. If the concentration of hydrogen peroxide is doubled the rate of reaction should double as well. The rate should steadily increase when more substrate is added because more of the enzyme’s active sites are being used which results in more reactions so the required amount of oxygen is made more quickly. Once the amount of substrate molecules added exceeds the number of active sites available then the rate of reaction will no longer go up. This is because the maximum number of reactions are being done at once so any extra substrate molecules have to wait until some of the active sites become available.
To test out how the concentration of hydrogen peroxide affects the rate of reaction this is what has been done:
Grind up exactly 4g of potato with a pinch of sand (the amount of sand doesn’t need to be accurate because it doesn’t affect the results) in the pestle and mortar. Add this to the conical flask.
Use the measuring cylinder to measure out 25cm3 of hydrogen peroxide the 100% concentration. It is important to measure the amount accurately to ensure the experiment is impartial.
Prepare the water bath by using hot water from the tap and altering it using some cold water until the range (40-45oC) is reached.
Pour the hydrogen peroxide into the conical flask and immediately insert the bung, at the same time start the stopwatch.
Bubbles should start to rise up the conical flask and fizzing will be noticeable. The gas syringe will start to move quite rapidly.
Shake the conical flask vigorously before every reading is taken.
Recordings will be taken from the gas syringe every 30 seconds.
When the reaction has finished wait for approximately 2 minutes to see if any more oxygen evolves then stop the stopwatch.
Repeat the experiment twice more for that concentration and note the results.
Repeat the experiments 3 times for each concentration 80%, 60%, 40%, 20% and 0%, repeating the experiments will help to produce more accurate results as any inaccuracies in one experiment can be compensated for in the other experiments.
The 0% concentration of hydrogen peroxide is used as a control to show that at 0% concentration no reaction occurs.
To be protected safety goggles must be worn at all times during the experiment. A lab coat should be worn to protect from damage to clothing. Cleaning apparatus must be kept near by in case of any spillage.
Hydrogen peroxide is toxic and care must be taken when using it because it will blister the skin easily.
Time (s)Experiment 1Experiment 2Experiment 3AverageRate of reaction
Time (s)Experiment 1Experiment 2Experiment 3AverageRate of reaction
Time (s)Experiment 1Experiment 2Experiment 3AverageRate of reaction
Time (s)Experiment 1Experiment 2Experiment 3AverageRate of reaction
Time (s)Experiment 1Experiment 2Experiment 3AverageRate of reaction
The replicates are all written down to the nearest whole one on the gas syringe, because sometimes the gas syringe was moving so fast it could not be recognised if it was at .5 of a cm3 .
From these results it was able to plot a graphs of oxygen evolved and rate of reaction.
It would have been expected that when the concentration doubled so would the rate of reaction but this didn’t occur. After 270 seconds the rate of reaction slows almost to a halt. The straight lines show this on the graphs. At this point virtually all of the active sites are occupied so the active sites are said to be saturated with hydrogen peroxide. Increasing the concentration at this point will not cause the rate of reaction to go up any more. . All the active sites are being used so any extra Hydrogen Peroxide molecules will have to wait until an active site becomes available.
The theoretical maximum rate of reaction is when all the sites are being used but 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. The time taken to fit into and leave the active site is the limiting factor in the rate of reaction.
There is something obviously wrong with the results because the rate of reaction should be highest at 100% concentration.
These could be some of the explanations to illustrate why the experiment went wrong
1. The experiment was repeated 3 times to and an average was taken improve accuracy. The results were used to plot graphs with a lone of best fit. It was unrealistic to think that all the variables could be kept the same.
2. There was a slight delay between pouring the hydrogen peroxide into the conical flask, inserting the bung and starting the stopwatch. This will slightly affect the results but the three steps were carried out in the same way for each experiment so it shouldn’t have made a very big difference in the overall results.
3. One of the most inaccurate pieces of apparatus used was the measuring cylinder this is extremely inexact because if the meniscus isn’t exactly on the line the measurement is wrong. This brings in a fairly large percentage error.
4. The volume of gas in the conical flask is very slightly affected by how far the bug is pushed down in each experiment, if the bung is pushed down further then the volume in the conical flask will be less so more oxygen will be pushed into the gas syringe early in the experiment and this might have affected the results.
5. The gas syringe only had measurements for 1 whole number this could have affected the results because no decimal places could be used.
6. The amount of catalase in each potato couldn’t be measured this would affect the results because some parts of the potato could have more catalase than the rest.
7. The water bath was difficult to keep at the constant temperature due to heat loss to the surroundings more hot water would have to be added to heat it up again but this is imprecise.
8. The hydrogen peroxide could have been at different temperatures due to the room warming and cooling.
9. The potato ran out so another potato was used this could have influenced the results because this potato could have contained a different concentration of catalase.
10. The balance only measures to 2 d.p. this might have affected the results because slightly different masses of potato could have been used.
11. The gas syringe may have been slightly sticky making it harder for the oxygen to push, to register the true result.
1. To gain a more accurate result more replicates could be taken to get a more actual average.
2. There is no way to rectify the time taken between pouring in the hydrogen peroxide, putting in the bung and starting the stopwatch. Although it does help if there are assistants to help so each person only has to do one job.
3. Instead of using a measuring cylinder a pipette or a burette would have been far more precise pieces of equipment.
4. A conical flask with a pressure line could have been used this would show how far to push in the bung.
5. A decimal place gas syringe would give more accurate readings.
6. The catalase can’t be measured in potato a much better catalase substitute would be yeast this can be weighed and measured. Also once the potato is used another would have to be used and this could produce catalase of a totally different concentration.
7. An electrically monitored water bath would be useful this could keep the water in the correct temperature range accurately.
8. The hydrogen peroxide should be kept at a constant temperature by putting it into an electrically monitored water bath and taking and recording its temperature before it is added to the catalase.
9. A different source of catalase should be used one that’s concentration can be measured e.g. yeast.
10. A balance with more decimal places would have made the results more exact.
11. The gas syringes should be made so that it has a non-stick surface inside so it doesn’t become stiff.
The plotted results on the graph go up to produce a curve of best fit levelling off at the end. The main anomaly is the fact that the rate of reaction at 40% concentration is far higher than that at 100% concentration. Another one of the anomalies in the results was the 3rd experiment at 60% concentration this went completely wrong. These were probably due to and experimental error involving some of the limiting factors mentioned above.
