Catalase is an enzyme that catalyses the reaction by which hydrogen peroxide is decomposed to water and oxygen. 2H2O2 ⇔ 2H2O + O2
Found extensively in mammalian tissues, liver, muscle (beef), apple and potato. catalase prevents the accumulation of and protects the body tissues from damage by peroxide, which is continuously produced by numerous metabolic reactions. In humans hydrogen peroxide is catalysed by catalase in the liver. Hydrogen peroxide needs to be broken down because it is poisonous.
Preliminary experiment
I carried out a preliminary experiment in order to familiarise myself with the procedures of this experiment. To find flaws in the method which can be improved, in order to increase the accuracy and reliability of the results that will be collected for this investigation. I carried out an experiment that can be used to measure the volume of oxygen produced, when a range of hydrogen peroxide at different concentrations is catalysed by catalase. The expected results of an experiment like this can be seen in the graph below.
Errors and improvements
When carrying out the preliminary experiment I found faults in the method that can be improved. I encountered problems using a 100 ml measuring cylinder to measure the volume of gas produced. I encountered errors because it was not very accurate, due to its large diameter and the graduations being at large increments. Hence a large volume of gas may escape. There is also a large inbuilt error, which decreases the accuracy of the readings.
To overcome this problem I will use a burette to measure the volume of gas produced. A burette has a much smaller diameter and is graduated at smaller increments. Therefore as a result less gas will escape and more accurate readings will be obtained due to the decrease in inbuilt error.
The syringe being used to add the catalyse solution was inserted to the bung on the reaction chamber. This, however, had small gaps from where some of the gas may escape. This decreases the accuracy of the readings, because not all the gas produced is being measured.
To avoid this error when carrying out this experiment again I will put plasticine around the syringe and delivery tube. By doing this I can ensure that the top of the bung is air tight, and no gas is escaping. This will mean that the amount of gas escaping is decreased, therefore a more accurate reading of the actual volume of gas produced.
To get the required volumes of reactants I used a 100 ml measuring cylinder in the preliminary experiment. For the volumes of reactants I was using this was inappropriate, because the graduations were very large for this. Therefore as a result the accuracy of the measurements were very limited and would not be appropriate to use for this experiment.
To resolve this error and increase the accuracy of the measuring instrument to an appropriate degree of accuracy. When I carry out this experiment again I will be using a 10 ml measuring cylinder. This will increase accuracy because 10 ml is the greatest measurement of reactant I will be taking. A 10 ml measuring cylinder is graduated at smaller increments; therefore it has a smaller inbuilt error and increases accuracy.
The temperature of the reactants in the preliminary was not kept constant. This may allow for fluctuations in temperature to affect the rate of results. If temperature increases the molecules gain kinetic energy and there are more successful collisions. Hence the results obtained from an uncontrolled reaction may occur due to an external factor.
To keep the temperature constant I will carry out the reactions with the reaction chamber in a water bath. I will use a water bath to maintain a constant temperature of 30°C in a beaker, throughout the experiment. This will ensure that all tests are carried out in the same conditions and the results are not obtained with the influence of an external factor.
These are improvements that I will make when carrying out this experiment again. I have made these changes in the method to increase the accuracy and reliability of the results obtained. This will enable me to calculate and analyse the results more efficiently and effectively.
Preliminary Results
Results at 200C
Result at 300C
Results at 400C
Yellow = 40 degrees. Blue = 30 degrees. Pink = 20 degrees.
Prediction
The aim of this investigation is to investigate how the substrate concentration affects the activity of catalase on hydrogen peroxide. I will do this by carrying out an experiment in which I will measure the volume of gas produced at when catalase kept at a constant concentration is used to catalyse different concentrations of hydrogen peroxide.
I predict that as the concentration of hydrogen peroxide is increased so will the rate of reaction. I think this will happen until a point whereby all the active sites are in use. At this point the rate of reaction will plateau and become constant.
When the concentration of an enzyme is kept constant, the substrate concentration is varied, the rate of reaction is affected. At low substrate concentration there are few substrate molecules around. Therefore there are few successful collisions between substrate molecules and active sites. Few enzyme-substrate complexes are formed and the rate at which the substrate is catalysed is slow. This is also due to their being more active sites then substrate molecules, meaning that all of the active sites are not in use at any one time.
As the substrate concentration is increased, there are more substrate molecules around. Therefore there are more successful collisions and more enzyme-substrate complexes formed. As a result more product is being catalysed faster because a greater number of the active sites are being used. The rate of reaction is increased because more active sites are being used at any one time, therefore more product can be catalysed.
When the substrate concentration is high, at which point there are more substrate molecules than there are active sites. There are many successful collisions taking place and many enzyme-substrate complexes being formed. The rate of reaction at this point is at its optimum, because all of the enzyme’s active sites are being used at any one time. This is known as the Vmax, at which point an enzyme is working at its maximum possible rate. Substrate molecules at this point are queuing up for an active site to become free.
Increasing the substrate concentration above this point will have no affect on the rate of reaction. This is due to the number of active sites being the limiting factor, and an increase in rate of reaction would only occur if enzyme concentration is increased. Therefore I predict that as the concentration of hydrogen peroxide is increased, the rate of reaction will also increase. It will increase until the point where the enzyme has reached its Vmax and the rate of reaction becomes constant and plateaus. I predict that the enzyme will reach its Vmax at around 1.8–2.0 mol/dm3 concentration of hydrogen peroxide.
Variables
There are a number of variables that I will take into account when carrying out this experiment. Some of these are dependent; some independent, which will need to be maintained. The dependant variable in this experiment is the concentration of hydrogen peroxide. The results I obtain will be due to changing this variable. Controlled variables are ones that I will keep constant and carry out measures to maintain them. I must keep the independent variables constant in order to obtain a reliable and accurate set of results that display the effect changing the dependant variable has to this experiment. Below is a table of how these variables will affect the experiment and how it will be controlled.
Safety
These are the safety measures I will apply when carrying out the experiment to ensure that it runs smoothly.
- Wash your hands before and after the experiment
- Use care when working with glassware.
- Use care when working around electrical sources.
- Use care when using any chemicals in the laboratory.
- Use care when using the hydrogen peroxide to not inhale it.
- Make sure goggles are worn at all times.
- Make sure all laboratory surfaces are kept clean and clear throughout the experiment.
- Make sure loose clothing is removed or tucked in.
- Make sure long hair is tied back.
- If there are any breakages or spillages inform the laboratory staff in charge, and take appropriate course of action under their direction.
Apparatus
Dilution table
In order to obtain accurate concentrations of hydrogen peroxide I have produced a dilution table. I will use it to dilute the given 2.0 mol/dm3 of hydrogen peroxide to the required concentrations.
Method
- Before starting this experiment ensure that the area being used to carry out the experiment is clear, and put goggles on.
- Collect and rinse out the measuring instruments being used with the solutions that are going to be used with them, to remove any substances remaining from previous usage.
- Set up the apparatus, ensuring that the burette is straight, and the delivery tube is going to the burette. Use plastecine to make sure that the bung with the delivery tube is airtight.
- Put six boiling tubes in each boiling tube rack.
- Using the glassware marker label the range of hydrogen peroxide concentrations on the boiling tubes.
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Dilute the hydrogen peroxide solution, by using a measuring cylinder to add required volumes of 2.0 mol/dm3 concentration of hydrogen peroxide solution and distilled water into the corresponding boiling tube. Starting with the lowest concentration first that is 0.4 mol/ dm3 of hydrogen peroxide solution.
- As each dilution of hydrogen peroxide has been made bung the boiling tube to seal it, to make sure the hydrogen peroxide doesn’t evaporate.
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Starting with the lowest concentration of hydrogen peroxide solution. Remove the bung and pour 3 cm3 of catalase into the reaction chamber, and quickly close it with the bung that has the delivery tube.
- Record the initial reading on the burette and start timing the test using the stopwatch.
- At the specified times read off the volume of water on the burette and record in the results table.
- Once the duration of the experiment is over do not replace bung rubber bung on boiling tube, this is dangerous as the reaction may still be carrying on.
- Repeat this for the other concentrations of hydrogen peroxide solution.
- Clear away all the apparatus.
- Calculate the volume of gas produced, the average gas produced at each concentration, and the rate of reaction for. Record values in results the table.
Reliability
I think that this method is accurate and reliable, due to the accuracy of the apparatus being used. Which allows for precise measurements of the volume of gas produced to be taken. Measurements will be taken by the use of a burette to 2 decimal places. I will make sure that that the burette is straight at 90 degrees to the work surface at all times, to avoid parallax error.
In addition to this other fair testing measures will be maintained throughout the experiment to limit errors in the results. One of these fair testing measures is that each concentration of hydrogen peroxide solution will be repeated so that two sets of results will be taken. In total twelve samples of the volume of gas produced will be taken. This will allow me to calculate the mean average of the two sets of results and give me a more reliable set of results.
To keep the dilutions of hydrogen peroxide remains at the required concentrations until use. I will cap the boiling tubes with a rubber bung once the dilution has been made. This will mean that the hydrogen cannot react and form water, or evaporate. As a result decreasing the concentration of the hydrogen peroxide and inaccurate results. The use of a bung to seal the boiling tubes until the solution is required will prevent this occurring and the results will be more accurate and reliable.
To allow consistency I will use the same measuring cylinder throughout, one to measure distilled water and the other to measure hydrogen peroxide solution. This will mean that all the measurements are subject to the same inbuilt error, and not to different ones if more than one measuring instrument was to be used and both had different inbuilt errors. As a result I will be limiting the error when measuring the solutions.
I will use a separate measuring cylinder for distilled water and hydrogen peroxide solution to avoid contamination. When using hydrogen peroxide solution I will start with the lowest concentration first. This will ensure that remainders of higher concentrations of hydrogen peroxide do not increase the concentration of the more diluted solution.
Another measure is when using the measuring cylinder I will make sure that all measurements are read off at exactly eye level. I will also use a stopwatch to time the duration of the experiment. This will allow for the duration of 180 seconds to be timed exactly.
Finally, I will carry out all the tests in the same place. This will mean that all the samples will experience the same conditions. Rather than an experiment whereby one set of tests being carried out in different conditions, which would result in unreliable and inaccurate results.
These measures that I will maintain throughout the experiment will allow for an accurate and reliable set of results. Some of which have been introduced as improvements to the errors in the preliminary experiment. Overall, I think that this method will be carried out to an appropriate degree of precision, allowing for a set of accurate and reliable set of results to be obtained.
References
- Biology 1, Cambridge University Press, 2002
- Britannica CD 97
- Biology, John Parker, 2000
Results table showing the volume of oxygen collected (cm3) when hydrogen peroxide is catalysed by catalase
Results table showing the volume of oxygen collected (cm3) when hydrogen peroxide is catalysed by catalase
Table showing the rate of reaction (cm3 O2min-1)
I will display the rate of reaction at 30 seconds for each substrate concentration in the form of a line graph. In order to get an accurate line of best fit I will use regression to find the equation of the best-fit line.
The equation of the line of regression line of y on x is:
y = α + βx
Where β = Sxy = ∑xy - ∑x∑y
Sxx n
∑x2 - (∑x) 2
n
and α = y - βx
Rate of reaction (cm3 O2min-1)
x = 7.00 ÷6 y = 5.00 ÷ 6
= 1.17 = 0.83
β = 8.20 – 7 × 5 = 2.37 = 1.45
6 1.63
9.80 – (7) 2
6
α = 0.83 – (1.45 × 1.17) = -0.87
Line of regression: y = 1.45x – 0.87
Analysis
The graph showing the average volume of oxygen collected when hydrogen peroxide is catalysed by catalase shows a general trend. As the concentration of hydrogen peroxide is increased the volume of oxygen produced also increases. At a low concentration of hydrogen peroxide 0.4 mol/dm3 an average of 1.10 cm3 of oxygen was collected after 180 seconds. From the graph it is also evident that the reaction doesn’t begin until after 30 seconds.
At this low concentration of hydrogen peroxide the reaction is slow, because there are few substrate molecules around. Therefore there are few successful collisions between substrate molecules and active sites. Few enzyme-substrate molecules are formed and the rate at which the substrate is catalysed is slow. The slow rate of reaction is also due to their being more active sites than substrate molecules, meaning that all of the active sites are not in use at any one time.
At 1.0 mol/dm3 concentration of hydrogen peroxide solution the volume of oxygen collected is 1.6 cm3. From the graph you can also see that this reaction started as soon as the reactants were mixed. There is an increase in oxygen collected, because there are more substrate molecules around. Therefore there are more successful collisions and more enzyme-substrate complexes formed. As a result more product is being catalysed faster because a greater number of the active sites are being used. The rate of the reaction is increased because more active sites are being used at any one time, therefore more product can be catalysed.
The highest concentration of hydrogen peroxide used was 2.0 mol/dm3, which produced an average of 2.15 cm3 of oxygen after 180 seconds. The substrate concentration is high and therefore a large number of substrate molecules are present. As a result there are many successful collisions taking place and many enzyme-substrate complexes being formed.
The general trend of the results can also be seen on the graph showing the rate of reaction (cm3 O2min-1) at 30 seconds for different concentrations of hydrogen peroxide. The graph shows that the rate of reaction at 30 seconds is increasing as the concentration of the hydrogen peroxide increases. The lowest rate of reaction is 0.00 cm3 O2min-1, which occurs at 0.4 mol/dm3 concentration of hydrogen peroxide. As the concentration of hydrogen peroxide increases, the rate of the reaction also increases in a linear trend.
Overall, I can conclude that the results displayed on the graphs show the predicted trend. As the concentration of hydrogen peroxide increases, the rate of reaction also increases. This is due to more substrate molecules being around to collide successfully to form enzyme-substrate complexes as substrate concentration increases.
Evaluation
I carried out the experiment as I planned in the method, however some procedures in the planning were not feasible practically or were changed by laboratory staff. After carrying out this experiment, I can say that the method used was adequate to produce a set of results that showed a general trend. It produced adequate results as I obtained two sets of readings for each concentration of sucrose solution being tested. The results were also reliable as I ensured fair testing measures were applied throughout the experiment.
The adequate results obtained I obtained, contain anomalous results. These anomalous results may have occurred due to flaws in the procedures, which may have been accounted for in the planning, but practically were not as expected.
These anomalous results can be seen on the graph that displays the average volume of oxygen collected when hydrogen peroxide is catalysed by catalase. One anomaly occurred at 0.8 mol/dm3 concentration of hydrogen peroxide solution. The other anomaly occurred at 1.2 mol/dm3 concentration of hydrogen peroxide solution.
These anomalies may have occurred as a result of minor errors. The effects of these errors have been only on these results and have not had a constant impact on the whole experiment.
One of these minor errors may have been that the concentrations of hydrogen peroxide solution gave anomalous results. When placed in the boiling tube rack these concentrations may have been exposed to greater sunlight and an increase in temperature. Therefore may have caused them to have greater kinetic energy, and hence increased the number of successful collisions, and the amount of product formed.
The minor errors above may have been a cause of the anomalous results in this experiment. These errors however were not the only errors of this experiment that had an impact on the results obtained. There were also major errors that were constant throughout the experiment and had an impact on all of the results. These major errors occurred due to some of the procedures in the planning not being feasible practically or were changed by laboratory staff. Below is a table of these major errors and the improvements I would make to resolve them.
Overall, after carrying out this experiment I have obtained an adequate and reliable set of results. The method used to carry out the experiment had some flaws in some of the procedures that were not feasible practically or were changed by laboratory staff. I have made improvements to these major errors that occurred to resolve them. These improvements were made to ensure that a more accurate and reliable set of results will be obtained, if I was to carry out this experiment again.