This is an experiment to examine how the concentration of the substrate Hydrogen Peroxide (H2O2) affects the rate of reaction of the enzyme Catalase.
Effect of Substrate Concentration on Catalase.
Aims
This is an experiment to examine how the concentration of the substrate Hydrogen Peroxide (H2O2) affects the rate of reaction of the enzyme Catalase.
Background Information
Enzymes such as Catalase are protein molecules, which are found in living cells. They are used to speed up specific reaction within the cell. They are all very specific as each enzyme just performs one particular reaction.
Catalase is an enzyme found in food such as potato and liver. It is used for removing Hydrogen Peroxide from cells. Hydrogen Peroxide is the poisonous by-product of metabolism. Catalase speeds up the decomposition of Hydrogen Peroxide into water and oxygen as shown in the equations below.
Formula
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.
Apparatus Need For The Experiment.
. 250 cm3 Glass Beakers
2. Glass Thistle Funnel
3. Graduated Measuring Cylinder
4. Cork Borer
5. 250 cm3 Glass Cylinders.
6. Digital Stop clock
7. Scalpel
8. Tap and Distilled Water
9. Plastic rule.
0. Safety Goggles.
Method
To test out how the concentration of Hydrogen Peroxide affects the rate of reaction first set up the apparatus and prepare the different concentrations of Hydrogen peroxide as below:
Concentration of Hydrogen Peroxide Volume of Hydrogen Peroxide (cm3) Volume of Tap Water (cm3)
25 vol 250 0
20 vol 200 50
5 vol 150 100
0 vol 100 150
5 vol 50 200
. Using the cork borer, take a sample of potato, cut this piece in cm intervals using the scalpel and plastic rule. Then cut these a further 2 sections, so there is now 5 sections per 1cm piece of potato. Using distilled water; allow the potato discs to stick to the side of the glass Thistle Funnel.
2. Fill a beaker with about 4cm of the pre-made measured solution of Hydrogen Peroxide ...
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25 vol 250 0
20 vol 200 50
5 vol 150 100
0 vol 100 150
5 vol 50 200
. Using the cork borer, take a sample of potato, cut this piece in cm intervals using the scalpel and plastic rule. Then cut these a further 2 sections, so there is now 5 sections per 1cm piece of potato. Using distilled water; allow the potato discs to stick to the side of the glass Thistle Funnel.
2. Fill a beaker with about 4cm of the pre-made measured solution of Hydrogen Peroxide and using the graduated measuring cylinder, completely fill the 10 cm3 cylinder with the same solution.
3. Immerse the thistle funnel into the beaker with the steam below the surface of the Hydrogen Peroxide over the funnel stem.
4. Measure the volume of oxygen evolved in a time of 5mins.
5. Repeat the procedure with the other Hydrogen Peroxide solutions. Be aware not to contaminate each solutions with one another, this will cause inaccurate results.
6. Repeat all he tests at least three times (Time dependent) so that an average can be obtained. Repeating the experiments several times will help to produce better and more accurate results, as any inaccuracies in one experiment should be compensated for by the other experiments. Note all the results in a table such as the one below.
This gives the rate in cm3 of oxygen produced in the time of 5 minutes, this is because I am timing how much oxygen gas is given off in the space of 5 minutes for the various concentrations of Hydrogen Peroxide.
From these results, a graph can be plotted with concentration on the x-axis and the volume of gas released on the y-axis.
To ensure this experiment is completed as fairly as possible, all the variables except for the concentration of Hydrogen Peroxide must be kept the same for all experiments. Variables that must not be altered include:
Temperature, Catalase Concentration, dimensions of potato, air pressure and humidity.
When measuring the volumes of water and Hydrogen Peroxide, the measurement should be taken from a 90-degree angle to avoid parallax error.
Predictions
I predict that as the substrate concentration increases, the rate of reaction will go up at a directionally proportional rate until the solution becomes saturated with the substrate Hydrogen Peroxide. When this saturation point is reached, then adding extra substrate will make no difference.
The rate steadily increases when more substrate is added because more of the active sites of the enzyme are being used which results in more reactions so the amount of Oxygen released in a given time is higher. 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 due to the maximum number of reactions being done at once, so any extra substrate molecules have to wait until some of the active sites become available.
Results
I carried out the above experiment and these results were obtained.
H2O2 Conc. (Vol) 25 Vol 20 Vol 15 Vol 10 Vol 5 Vol
O2 Released (Vol) - First 2.00 1.84 1.46 1.11 1.00
O2 Released (Vol) - Second 2.03 1.86 1.50 1.13 0.8
O2 Released (Vol) - Third 2.02 1.86 1.47 1.11 0.90
Average of the Tests 2.0 1.90 1.5 1.1 0.9
The average results are all written down to one decimal place because it is very hard to judge the gradient of a glass cylinder.
Interpretation
When the concentration of Hydrogen Peroxide is increased, the rate of reaction increases at a directionally proportional rate until the concentration of Hydrogen Peroxide reaches about 10vol. If the concentration were doubled, I would expect the amount of Oxygen released to be a figure twice as much.
From 25vol to 10vol shows a directionally proportional decrease in reactivity rate, after 10vol the rate of reaction slows down. This is shown by the gradient on the graph going down. At this point virtually all active sites are occupied making the active sites saturated with Hydrogen Peroxide. With an increase the concentration of Hydrogen Peroxide, the number of active sites increases, hence, makes a more violent reaction (Quicker).
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 active sites are being used at the same 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.
Limitations
To enable this experiment to be completed as accurate as possible, I repeated it three times and then used and then used an average of all the results to best plot a graph with a line of best fit. I tried my best to keep all the variables apart from the one I was testing (Hydrogen Peroxide Concentration) the same.
However and unfortunately in practice it is impossible with the basic apparatus I had to keep all measurements precisely the same. For example:
. There is a slight delay between pouring the thistle funnel with the potato discs into the beaker of Hydrogen Peroxide. This will slightly affect all the results for each individual experiment but as I carried out all the steps in the same way, it should not make any negotiable difference to the overall result.
2. It is also impossible to precisely measure out the amounts of Hydrogen Peroxide and Distilled Water each time. As the scale on the measuring cylinder shows the measurement to the nearest 1mm3, the solutions that I used should be correct to the nearest mm3.
Criticisms & Ways to Improve Experiment.
As using catalase founded in potatoes, the desired amount was hard to measure, however, measuring the amount of potato wasn´t difficult although that piece could contain different amounts of catalase compared to another piece. The results that would show from both pieces would be negotiable.
Maybe having a source of catalase from yeast would have been much more easier to use and handle. Specific amounts could be measured much more precisely.
