- 1 ruler
- 1 test-tube rack
- 1 bottle hydrogen peroxide
- 1 electronic weighing scale
- 1 measuring cylinder
- 1 Bunsen burner
- 1 250ml beaker
- 1 crucible and grinder
- 1 white tile
- Distilled water
- 14 identical test-tubes
- Matches
Method:
1. Weigh and prepare 7 2g samples of potato using an electronic scale.
2. With a grinder, grind the samples separately in the crucible and set each 2g sample on a white tile.
3. Pour out 10cm3 of hydrogen peroxide into each test-tube using a measuring cylinder and place them on a test-tube rack.
4. Leave 4 test-tubes in the freezer for 45 mins.
5. Half-fill the beaker with water and, placing one test-tube of hydrogen peroxide into the beaker, heat the liquids over a Bunsen flame. Using the thermometer, monitor the temperature of the hydrogen peroxide in the test-tube.
6. Turn off the flame when the hydrogen peroxide is heated to 25˚C.
7. Immediately add one 2g sample of potato into the test-tube and record your observations of any activity and measure along the outer side of the test-tube the height of the foam/bubbles that forms in the next 5 minutes.
8. Heat another test-tube of hydrogen peroxide to 25˚C but observe for 5 minutes without adding any potato. Record your observations.
9. With another test-tube, repeat steps 5-8 but instead of heating the hydrogen peroxide to 25˚C, heat to 35˚C.
10. With another test-tube, repeat steps 5-8 but instead of heating the hydrogen peroxide to 25˚C, heat to 45˚C.
11. With another test-tube, repeat steps 5-8 but instead of heating the hydrogen peroxide to 25˚C, heat to 55˚C.
12. With another test-tube, repeat steps 5-8 but instead of heating the hydrogen peroxide to 25˚C, heat to 65˚C.
13. 45 mins after leaving them in the freezer, take out the 4 test-tubes.
14. With two of the test-tubes of cold hydrogen peroxide, repeat steps 5-8 but instead of heating the hydrogen peroxide to 25˚C, heat to 5˚C.
15. With the remaining two test-tubes, repeat steps 5-8 but instead of heating the hydrogen peroxide to 25˚C, heat to 5˚C.
16. Organise your data into a results table.
Temperature, being the independent variable, was varied in the experiment to see if enzyme activity would be affected by the amount of heat supplied. This was accomplished through the use of a Bunsen flame and freezer, allowing for temperature to be monitored and controlled at regular intervals.
The changes of the dependent variable, the amount of enzyme activity occurring, caused by variations to the independent variable were collated at every 5 minutes. This was measured by the height of foam/bubbles produced as a result of the reaction catalysed by catalase in the potato, as observed from a previous experiment involving the same reactants and products. The greater the height of foam/bubbles, the greater the amount of enzyme activity could be inferred to have taken place.
To control all other variables apart from temperature, hydrogen peroxide from the same bottle was used, potato samples from the same potato were experimented with and ground to approximately the same extent. It was also important to check that all test-tubes were of similar make as different sized test-tubes might have openings with different radii, thus affecting the volume of foam/bubbles in 1 measured centimetre; a test-tube with a greater radius would contain more foam/bubbles than one with a smaller radius upon measuring 1cm along the side of the test-tube.
Data Collection
Table 1.1: Summary of quantitative data.
Table 1.2: Summary of results detailing height of foam/bubbles produced from approximately the same mass of potato across a range of temperatures.
Regardless of the temperature at which the hydrogen peroxide samples were, all trials were observed to have a similar reaction when potato samples were added into the test-tube. Bubbles formed around the potato and the hydrogen peroxide became milky with foam; these effects were observed in a short space of time at 25˚C and 35˚C and longer towards the extreme temperatures of 5˚C and 65˚C. There were no reactions observed in the test-tubes without potato samples in the experimented range of temperatures.
Data Processing and Presentation
Graph 2.1: Relationship between temperature of substrate (independent variable) and height of foam/bubbles and therefore the amount of enzyme activity (dependent variable).
Table 2.2: Calculations comparing proportions of percentage change in amount of enzyme activity as temperature increases.
Graph 2.2: Relationship between temperature increase and the proportion of percentage change in amount of enzyme activity.
Table 2.3: Calculations showing average rate at which foam/bubbles formed
Graph 2.3: Relationship between temperature and the average rate of formation of foam and bubbles, detailing enzyme activity.
Conclusion/evaluation
My hypothesis that enzyme activity in plant cells would be low at low temperatures and increase as temperature increases until temperatures reach 37˚C, beyond which there will be a rapid loss of activity, has been somewhat supported with reference to the results of my experiment. Although the temperature of optimum enzyme activity, denoted by the greatest amount of foam and bubbles produced, was not 35˚C as expected but 25˚C, the basis of my hypothesis that has been proven; the lower temperatures (5˚C, 15˚C) had less enzyme activity than that at 25˚C, and the higher temperatures (35˚C, 45˚C, 55˚C, 65˚C) witnessed rapidly decreasing rates of enzyme activity.
However, there were limitations in the procedure of the experiment which inevitably affected my results. Firstly, 5 minutes would be too short a time frame to accurately observe the amount of enzyme activity. This is because the reactions between catalase and hydrogen peroxide did not end in 5 minutes, so the data obtained from my experiment do not reflect the total amount of enzyme activity that would have taken place if the reaction was allowed to complete. Experimenting with a longer period of time could perhaps yield different results if there is a possibility that the rate at which catalase functions at a set temperature is not determined in this investigation and could fluctuate. To improve on this aspect of the experiment, time taken for the reaction to complete, judged by the discontinuation of foam/bubble production, can be investigated as this would provide a holistic picture of the total time required for reaction at a certain temperature. This would give me a more accurate result on rather than basing an analysis on 5 minute samples.
Also, the hydrogen peroxide-potato reactions were monitored without the use of equipment that regulated temperature, nor were the temperatures of the hydrogen peroxide at the end of every 5 minute-trial verified. In hindsight, this could have resulted in a change in temperature over the 5 minutes of each trial as there was ample time for the hydrogen peroxide to cool (if it had been heated over the Bunsen flame) or become warmer (if it was put in the freezer). A change in temperature would have affected my results; as temperatures changed towards the optimum of 25˚C (room temperature), the rate and amount of enzyme activity in 5 minutes would have increased as well, rendering my results inaccurate. This can be improved by using a water bath to maintain the temperature of the hydrogen peroxide. In this way, error resulting from the change in temperature can be effectively minimised, if not eliminated, and data obtained accurately reflect the amount of enzyme activity at each temperature.
Although the measuring of the level of foam/bubbles produced provides enough information to generalise a trend between temperature and the amount of enzyme activity, there are a number of weaknesses in this approach. Firstly, if other products are evolved (e.g. in the form of gas which does not convert to foam or bubbles), they would not be taken into account with the method of measuring the height of foam and bubbles. The under-measurement of products formed would be reflected in data processing of the investigation as a lowered amount of enzyme activity at a set temperature, thus affecting the observation used to generalise a trend or make future predictions. This limitation can be rectified with the modification of the method; to use a gas syringe to record the total volume of gaseous products formed. This includes gas from popped bubbles and foam, as well as gas which does not convert into bubbles and foam. Therefore, a better notion of total products obtained from the reaction can be explained, allowing for a more accurate comparison of evaluation of results to my initial hypothesis.
With reference to Graph 2.1, the trend of enzyme activity increasing with temperature until an optimum of 25˚C, only to fall rapidly at temperatures higher than this can be established. This is further substantiated by the compound percentage change in enzyme activity as seen in Table and Graph 2.2 as the percentage of reduction in the rate of enzyme activity falls with the increasing temperatures. The controls served in qualifying that as there were no reactions observed in the test-tubes without potato samples in the experimented range of temperatures, temperature is therefore deduced to have an effect on the rate at which the enzyme activity was carried out.
The effects of varying temperature on the rate of enzyme activity in potato cells has been determined by measuring the amount of foam and bubbles produced and offers some insight into the amount of enzyme activity. My hypothesis that enzyme activity in plant cells would be low at temperatures lower and higher than an optimum has also been supported to an extent. This has been accomplished by varying the independent variable (the temperature of hydrogen peroxide) to influence the dependent variable (amount of enzyme activity) while maintaining the controlled variables in an attempt to make this a fair experiment.
