Effect Of Substrate Concentration On The Activity Of Catalase

[Aparatus picture not reproduced]

. Add 2cm3 of yeast to one test tube. Add 4cm3 of hydrogen peroxide solution at a concentration of 20% to the other test tube. Use a pipette to measure out the volumes. It is very important to accurately measure the amounts of Hydrogen Peroxide, Yeast and water to ensure a fair test.

2. Pour the hydrogen peroxide solution into the test tube containing the yeast and immediately put the gas syringe bung on the end of the test tube, at the same time start the stopwatch.

3. Bubbles should start to rise up the tube and the gas syringe will move outwards, as soon as the gas syringe passes the 30cm3 mark stop the stopwatch and note the elapsed time down to the nearest 1/10th of a second.

4. Repeat the experiment with hydrogen peroxide concentrations 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 of Hydrogen Peroxide are made by adding tap water to the 20% Hydrogen Peroxide in the correct amounts. The table below shows what amounts of Hydrogen Peroxide and water are needed to make the solutions.

Concentration Of Hydrogen Peroxide

Volume Of Hydrogen Peroxide (cm3)

Volume Of Water (cm3)

20%

4

0

6%

3.2

0.8

2%

2.4

.6

0%

2

2

8%

.6

2.4

4%

0.8

3.2

0%

0

4

5. Repeat all the tests at least three times 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.

Hydrogen Peroxide Concentration

0%

4%

8%

0%

2%

6%

20%

Time Taken (Test 1)

Time Taken (Test 2)

Time Taken (Test 3)

Average of the Tests

Rate

The rate can then be worked out by

Rate=30/Average Time

This gives the rate in cm3 of oxygen produced per second, this is because I am timing how long it takes to produce 30cm3 of oxygen. From these results a graph can be plotted with concentration on the x-axis and time taken on the y-axis.

I am using yeast catalase as opposed to catalase from apples, potatoes or liver because it is easier to get the desired amount of yeast catalase by simply measuring it off. To obtain catalase from a substance such as potato would involve crushing it and with that method you would never be sure of the concentration of the catalase. If the catalase was used up then another potato would have to be crushed and this could produce catalase of a totally different concentration which would lead to inaccuracies in the experiment making this an unfair test.

To ensure this is a fair test all the variables except for the concentration of Hydrogen Peroxide must be kept the same for all the experiments. Variables that must not be altered include:-

Temperature, yeast concentration, type of yeast, batch of yeast, volume of yeast, volume of hydrogen peroxide, air pressure and humidity.

When measuring the volumes of Hydrogen Peroxide, Yeast and Water the measurement should be taken by looking at the scale at an angle of 90 degrees to it to avoid any parallax error.

RESULTS

I carried out the above experiment and these results were obtained.

Hydrogen Peroxide Concentration

0%

4%

8%

0%

2%

6%

20%

Time Taken (Test 1)

47.3

8.4

7.3

4.5

0.6

9.7

Time Taken (Test 2)

43.3

9

6.7

4.9

1.2

0

Time Taken (Test 3)

52.2

7.2

8.5

1.2

8.6

7.8

Average of the Tests

47.6

8.2

7.5

3.5

0.1

9.2

Rate=30/Average (Cm3/second)

0

0.63

.65

.71

2.22

2.97

3.26

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=30/Average Time

From these rates I was able to plot a graph of the rate of reaction against concentration of Hydrogen Peroxide.

CONCLUSION

When the concentration of Hydrogen Peroxide is increased, the rate of reaction increases at a directly proportional rate until the concentration of Hydrogen Peroxide reaches about 16%. If you double the concentration of Hydrogen Peroxide then the rate of reaction doubles as well. When the concentration is doubled from 8-16% the rate goes up from 1.65-2.97 Cm3 Oxygen produced per second, which is an increase of 1.8 times. I would expect the rate to increase two times if the Hydrogen Peroxide concentration is increased two times because there are twice as many substrate molecules which can join onto the enzymes active sites. The reason that the number is less than two times could be put down to the fact that at 16% the Enzyme's active sites may already be close to being saturated with Hydrogen Peroxide. There may also be some experimental error which causes the inaccuracies.

After 16% the increase in the rate of reaction slows down. This is shown by the gradient of the graph going down. At this point virtually all the active sites are occupied so the active sites are said to be saturated with Hydrogen Peroxide. Increasing the Hydrogen Peroxide Concentration after the point of saturation has been reached 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.

The diagram below shows what happens.

EVALUATION

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. However, in reality it is impossible to keep all the variables precisely the same. For example:

a) There is a slight delay between pouring the Hydrogen Peroxide into the yeast, putting the bung on and starting the stopwatch. 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. The volume of gas in the test tube to start with is slightly affected by the amount which the bung is pushed down each time, if the bung is pushed down further then the volume in the tube will be less so the 30cm3 of gas is reached faster.

c) Due to the fairly slow speed of our 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.

ANOMOLIES

The plotted results on the graph produce a straight line of best fit to begin with which then goes into a curve of steadily decreasing gradient. The only anomalies are the results at 8% and 10%. The result at 8% is slightly above the line of best fit and the 10% result is slightly below it. This is probably due to an experimental error involving one of the factors mentioned above.

EXTENSION

This experiment could be improved in a number of ways. It 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.

Using more concentrations of Hydrogen Peroxide would have produced a better looking graph and I would have liked to use concentrations higher than 20% to extend the graph so that the maximum possible rate of reaction could be reached.

The problem of the delay between pouring in the Hydrogen Peroxide, bunging the test tube and starting the stopwatch could have been limited by getting another person to start the stopwatch when the hydrogen peroxide was poured into the tube.