Reliability
To increase the reliability of the experiment 3 repeats will be carried out with each tissue extract. The results of these will be compared. Mean values will be calculated for the time taken for the discs to rise for each tissue extract.
Control
A control experiment will be carried out which proves that intact catalase with the correct tertiary structure (shape) is required for the reaction to occur. At the end of the main experiment, a control experiment will be run. The procedure will be the same (including all volumes) except that the catalase will be replaced by boiled and cooled catalase.
No breakdown of hydrogen peroxide should occur as boiling will have denatured the enzyme. The disc should not float as no oxygen is produced.
Hazards
The main risk is injury caused by hydrogen peroxide coming in contact with the skin or eyes. This could cause chemical burns as it is a caustic substance. To reduce this risk, only a relatively dilute solution will be used throughout. Care will be taken when handling it and safety spectacles worn throughout. Any spills will be reported to the teacher and then cleared up promptly under supervision.
Results
Trends
The discs soaked in the liver extract rose to the surface in the shortest time with a mean of 7.3 s. The discs soaked in turkey breast took the longest time to rise to the surface with a mean of 38.0 s.
Reliability
There was overlap between range bars of results from the potato, celery and thigh tissues, hence the differences in mean are not reliable.
Accuracy
Some discs took longer to rise because they ‘rubbed against’ the side.
Improvements
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There is no temperature control, although it was monitored. If the tube of hydrogen peroxide were maintained in a 250C thermostatic water bath until just before the discs were added, it would be constant.
- The pH needs to be controlled, this would be done by adding a pH 7buffer to each tube.
Explanation –
When a paper disc soaked in tissue extract (catalase) is placed in hydrogen peroxide solution, the oxygen produced collects on the disc and causes it to float.
2H2O2 → 2H2O + O2
In this experiment discs soaked in liver tissue extract floated to the surface in the shortest time (mean 7.3 s) as the extract contained the highest concentration catalase. At high concentrations of catalase there are more catalase molecules and therefore more available active sites, which form more enzyme-substrate complexes with hydrogen peroxide molecules. Therefore more oxygen is produced, meaning more collects on the discs in a shorter time, hence it took less time for the disc to rise.
Discs soaked in turkey breast tissue did not contain much catalase and so took a long time to generate the oxygen required to make the discs float. This is because at low catalase concentrations there are fewer catalase molecules and therefore fewer available active sites, which form fewer enzyme-substrate complexes with hydrogen peroxide molecules. Therefore less oxygen is produced, meaning it takes longer for it to collect in sufficient quantities to enable the disc to float, hence it took more time for the disc to rise.
Conclusion
It was predicted that discs soaked in extracts of animal tissue would rise to the surface in a shorter time than those soaked in plant tissue as they would normally respire faster and hence need more catalase to break down the toxic bi-product hydrogen peroxide. This was true for liver and perhaps for turkey thigh muscle but the breast muscle tissue contained less catalase than all the others. The prediction was mostly supported however the wide spread of results around some of the means indicate that further experiments are needed before a more definite conclusion may be made.
Further Work
In further work it would be interesting to explore how the time taken for discs to rise (dependent variable) changes with pH. The independent variable would be pH, changed using pH 5, pH 6, pH 7, pH 8, pH 9, pH10 buffers.
The controlled variables would be:
- The concentration of tissue extract (enzyme source) would be 25%
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Volume and concentration of hydrogen peroxide solution (substrate) will be kept the same for each disc (20cm3 of the 2% solution)
- The time that the discs were left in tissue extract was kept constant at 30seconds using a stopwatch.
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Temperature – the whole experiment will be carried out at 20oC in a thermostatically controlled water bath.
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5cm3 of each pH buffer solution will be used.
Most body cells work best in a pH close to neutral, hence the time taken for discs to rise will be least at this pH. At a slightly lower or higher pH (6 and 8) the time taken for the disc to rise would be longer as the charges on active site and substrate will repel. At extreme pH the reaction may stop if ionic bonds break and the enzyme denatures.
