∙ 10cm³ measuring syringe
∙ 100 cm³ of 20% concentration hydrogen peroxide
∙ Potato
∙ Scalpel and tongs
∙ Tile
∙ Ruler
∙ Cork borer (1 cm diameter)
∙ Test tube rack
∙ 10 Test tubes
∙ Trough
∙ Water
∙ Delivery tube and bung
∙ Clamp
∙ Stopwatch
∙ 10cm³ measuring tube
∙ Knife
The equipment will be set up as follows:
Five cylinders, each of 2cm length, will be bored from a potato using a cork borer. Using a knife, one piece will be left whole, the next cut into two pieces, the third cut into three pieces, the fourth cut into four pieces and the fifth cut into five pieces, on a tile. 10 cm³ of hydrogen peroxide will then be put into a test tube (using a measuring syringe to ensure accuracy). I will make sure the syringe is held at an 180º angle to the hydrogen peroxide when it’s being measured out and no air bubbles are in the syringe to ensure accuracy. The potato piece is then added to the test tube and the bung will be secured on (see diagram). The stopwatch will also be started at this time. The potato and hydrogen peroxide should start to react and oxygen will pass up the delivery tube and into the 10 cm³ measuring tube. After every 30 seconds I will measure how much oxygen is present in the measuring tube. I will take recordings up to 3 minutes and present my results in a table. This experiment will then be repeated another four times, each time using different pieces of potato. E.g. second time using two pieces of potato, third time using three pieces of potato etc….
There are many variables, which if altered, will affect the experiment. To maintain a fair and accurate test the only variable that will be altered is the concentration of the catalase (by varying the surface area of the potato). The other variables will need to be kept constant. These are:
∙ Temperature
As the temperature of the hydrogen peroxide is increased, the quicker the reaction will become because the molecules have more energy so they move more vigorously. This increases the chances of a hydrogen peroxide molecule and an active site meeting and would therefore increase the rate of reaction. As the temperature of the enzyme is increased the rate at which it meets with the hydrogen peroxide increases. All enzymes have an optimum temperature. This is the temperature at which it works most effectively. Once an enzyme becomes hotter than it’s optimum temperature it becomes less efficient. This is because the enzyme molecules are becoming denatured. This means the enzymes have too much energy so they start to breakdown bonds on active sites that give it stability and shape. The shape of the active site is then changed and the hydrogen molecules would no longer fit and a reaction would not take place. To maintain a constant temperature I will ensure there is little or no contact between my hands and the potato/hydrogen peroxide. I can do this by using tongs. All the experiments will be conducted at room temperature.
∙ pH
Like temperature, enzymes also have an optimum pH. At this optimum pH the enzymes work best and variations either side of the pH scale denature the enzyme causing it to work less effectively. To ensure a fair test all experiment will be conducted using the same pH of hydrogen peroxide.
∙ Concentration of Hydrogen Peroxide
A change in the concentration may have an affect on the rate of reaction. If there isn’t sufficient hydrogen peroxide to fill all the active sites then an increase in concentration will increase the rate of reaction because there will be more hydrogen peroxide molecules to fill the active sites. A lower concentration will decrease the rate of reaction. To maintain a fair test the same concentration of substrate (20%) will be used. The same volume of hydrogen peroxide will also be used.
∙ Inhibitors
In this experiment, Inhibitors would compete with the hydrogen peroxide for the active site of the enzyme. Inhibitors would slow down the reaction because they would use up active sites that the substrate molecules could be using. In some cases the Inhibitors can permanently change the shape of the active site so the substrate molecule would be unable to use it. Inhibitors should not affect my experiment because none will be added.
To make the experiment more accurate I will repeat the experiment so I can gain two sets of results then take an average result, which I will use to plot a graph. This will help to eliminate anomalous results.
During the experiment I will wear safety goggles because hydrogen peroxide is corrosive. I will also take care while handling the knife.
I conducted a preliminary study, which showed me how much potato to use and the best concentration of hydrogen peroxide to use. Given the equipment, I believe the method I am using is the best because I will be able to collect and measure all the oxygen given off during the reaction. The method should give accurate results because all the products off the reaction will be collected and none will be lost.
Analyses
Referring to the graph ‘ A graph showing the effects…’ you can see that all the lines of best fit pass straight through the origin. This indicates that when no catalase is present no reaction takes place because there are no active sites for the hydrogen peroxide to react with. The fact that the line of best fit is straight shows that the surface area of the enzyme is directly proportional to the rate of reaction. All the gradients show a positive correlation, which means more gas is produced as the reaction has longer to take place. This is because the active sites aren’t destroyed and are therefore being re-used by hydrogen peroxide molecules.
From the graph you can see as the surface area is increased there’s more gas given off, which means the rate of reaction increased. This is because a greater surface area means there are more active sites available, so there will be more collisions between the hydrogen peroxide molecules and the active sites.
This means the first half of my prediction was correct. By comparing the increase in surface area to the increase in the rate of reaction you can see the rest of my prediction was also correct. By referring to appendix 3 you can see that in most cases the increase in surface area was equal to the increase in the rate of reaction.
E.g. when the gradient was 1.2 times larger, the surface area available was 1.2 times bigger. This is because the active sites available were 1.2 times more.
Evaluation
I believe the method used was generally the best possible, considering the equipment available. If I were too repeat the experiment I would change the sizes of potato pieces used. Next time I would use a piece with double the surface area then triple the surface area etc. This may not change the accuracy of the experiment but it would make my calculations easier to complete and would therefore reduce the chance of human error.
By referring to the graph you can see two separate results that are circled. These indicate anomalous results. They are anomalous because they show points were two separate experiments have produced the same result. This should not occur because one experiment should react faster than the other. You can also see that not many of the results fit fall on to the line of best fit. This is because there were limitations to my method. There is a delay between the time of adding the hydrogen peroxide to the catalase and putting the bung on the test tube. This allowed some gas to escape. There was also a delay between the start of the reaction and the starting of the stopwatch. This reduces the accuracy of the results. I believe these factors would significantly affect the accuracy of my experiment. It’s also impossible to measure out accurately the hydrogen peroxide because the syringe measures to the nearest mm³ however I don’t think this significantly affected the reliability of the results. Another reason for the anomalous results is the fact that there was an un even arrangement of cells on the surface of the potato which means some surface would have more enzymes available compared to other pieces. You can tell this because some bits were a slightly darker colour compared to others. I used a lot of the potato so I was comparing pieces from the middle with pieces from the edge. This would affect the experiment because there would be different concentrations of enzymes in the middle and edge of a potato.
To improve the experiment a gas syringe could be used however more substrate and more enzymes would need to be used, otherwise the syringe wouldn’t move a significant amount to make it affective. A gas syringe would make it easier and quicker to read the amount of gas produced. If another person helped with the experiment then they could be used to start the stopwatch at exactly the correct time, improving the reliability of the results. The experiment could also be repeated more times and a more accurate average could therefore be taken. This is because anomalous results would be cancelled out by the other sets of results.
