To Compare the Concentration of the Enzyme Catalase in Plant v. Animal v. Fungal Tissues

As-Level Biology Coursework -Investigation 1

To Compare the Concentration of the Enzyme Catalase in

Plant v. Animal v. Fungal Tissues

Plan

Catalase is a widespread enzyme, which breaks down the waste product of metabolism. Hydrogen peroxide (H2O2) is a metabolic waste product, which is toxic and must be removed by cells. Catalase is the enzyme used by cells to remove H2O2. The reaction is as shown below:

Catalase enzyme

H2O2 --------------------------- H2O + 1/2O2

Hypothesis

My hypothesis is that animal and fungal tissues will have a higher concentration of catalase than plant tissues.

Catalase is the enzyme that is used to break down H2O2, which is a metabolic waste product, therefore the higher the metabolic rate of an organism the more waste product it will produce, which therefore means the higher the concentration of catalase the organism would contain.

Metabolism is the collective term for all the chemical reactions of life. An organism's metabolic rate is the amount of energy used by the organism to maintain its living function through the chemical reactions of life. Therefore, how an organism obtains the products needed to maintain its living function would affect its metabolic rate.

Animals and fungi are different from plants in that they are heterotrophic whereas plants are autotrophic. This means that animal and fungi feed on organic matter obtained from other organisms. Plants on the other hand are autotrophic or 'self-feeding', this means that they absorb inorganic nutrients- such as CO2 and minerals with which to make their organic compounds.

These two different methods of obtaining organic compounds would have a large impact on the concentration of catalase. For heterotrophic organisms by obtaining their organic compound from other organisms would absorb more products, which they would be unable to use. Therefore they would a) produce more waste product and/or b) they would have to use up more energy in chemical reactions to convert these useless products into useful ones. In the first way the metabolic rate would increase due to the amount of energy needed to break down the waste product and in the second way the metabolic rate would increase due to the energy needed to convert the product. Therefore if the metabolic rate increases so would the concentration of catalase, since more H2O2 would be produce, which would need to be broken down. Autotrophic organisms, on the other hand, by producing their own organic compound would not have the above two factors to deal with or if they did, it would not be on the same scale as hetroterophic organisms. This is due to the fact that by being self-feeding they would not produce products they could not use, unless in small quantity as a by-product in a chemical reaction to produce the product they do need.

This investigation is to compare two different types of plant tissues against two types of animal and two types of fungi tissues, to see whether the hypothesis set out above is borne out by experimentation. The investigation will be carried out at room temperature and in pH7 conditions, obtain by using a pH7 buffer. The same amount of tissues, 5g, and substrate (H2O2), 5cm3, will be use throughout. Both these measures are to ensure that any variables that might affect the investigation are controlled throughout.

The volume of oxygen produce when catalase breaks down H2O2 will be measured at ...

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The volume of oxygen produce when catalase breaks down H2O2 will be measured at set time intervals. From this an initial rate of reaction will be worked out. The initial rate of reaction is an indication of the concentration of catalase. For the higher the concentration of catalase the faster the initial rate of reaction would be.

Whilst this experiment does not have any very serious ethical consideration attached to it, some people may not like the use muscle and liver tissue used as the animal tissues part of the experiment. However, since the experiment requires the testing of catalase concentration in animal tissue against that of plant and fungi, there is not much that can be done. The experiment, of course, could be change for people with strong objections to the use of animal tissues in experimentation, to just comparing plant against fungi tissues.

Risk Assessment

Hydrogen Peroxide

A mild irritant to skin and eyes in dilute quantities used. Should not be swallowed or allow to come into contact with eyes. If swallowed, wash out mouth with water, drink a glass or two of water and seek medical attention. If liquid gets into eyes, flood the eye with gently running water for 10mins and seek medical attention.

The same applies for pH7 buffer.

Care should also be use when handling either knife or scalpel.

Pilot Experiment

In order to become familiar with the apparatus that was to be used, two pilot experiments using a gas syringe was performed. In the first different pH buffers were used to find out the effect of pH on the enzyme. In the second different concentration of inhibitors were used to test the effect of inhibitors on enzyme. In both experiment the 5g of tissue was placed in the test tube along with in the first experiment different pH buffers (5cm3) and the second with a mixture of pH buffer and inhibitor (5cm3), wherein the amount of buffer and inhibitor was varied to give different concentration. Then the test tube was connected to the gas syringe. After this a syringe containing 5cm3 of H2O2 was added to the test tube and a stopwatch was started. The results were then recorded.

The pilot experiment also help to determine the amount of tissue, substrate (H2O2) and buffer, which would be used in the actual investigation.

Method

Firstly the tissue was cut using a knife or scalpel, and then the tissue was crushed in a pestle and mortar. The tissue was then weighed on a top-pan balance, after which 5g of the tissue was place into a test tube. Then 5cm3 of pH7 buffer was added to the test tube using a pipette. The apparatus was set up as above. Then 5cm3 of H2O2 was pull up into a 20cm3 plastic syringe, the syringe was then set up as shown above and the H2O2 was added to the test tube as the stopwatch was started. The amount of O2 gas produce was then recorded at set intervals (30, 60, 90, 120, 150, 180 seconds). The experiment was repeated until two sets of data had been obtained from all the different types of tissue (dried yeast, fresh yeast, potato, celery, muscle and liver).

The equipment that was used was chosen for a number of reasons. The gas syringe was used due to the fact that it was the precise and accurate apparatus available for the measurement of volume of gas produce in an experiment. The use of the plastic syringe and two hole bung in the test tube was the best and quickest method of ensuring a relatively airtight condition, so as to allow no oxygen to escape once produce, and therefore more accurate results.

Modification

During the first set of experiments, it was discovered that 5g of tissue was too much. The reaction was too strong, thereby causing liquid to bubble up the test tube and then into the gas syringe unless the gas syringe was disconnected from the test tube. If liquid entered the gas syringe it no longer functioned properly therefore the gas syringe had to be disconnected. This means that the experiment had to be restarted from the beginning. Therefore, it was decided that the investigation should continue with only 2.5g of tissue being used.

Results

Analysis

The result shows that there is a significant difference between the initial rates of reaction in fungi tissue and animal and plant tissues. The raw data shows that on average yeast produces a volume of oxygen of around 5cm3, compared to animals, which only produce around 2cm3, and plants, which produce around only 1cm3. This is reflected in the initial rates of reaction whereby fresh yeast tissue has an initial rate of reaction over 12 times faster than potato tissue and over 6 times faster than liver tissue. This, therefore, indicate that fungi have a higher concentration of catalase than animals or plants.

Whilst it was expected that plants would have a lower concentration of catalase than either animals or fungi, it would have been expected that animals being the more complicated organism would have a higher concentration of catalase than fungi. This is due to the fact that as the more complicated organism they would be expected to perform more chemical reactions, thereby using more energy and increasing their metabolic rate and catalase concentration. However, as the results show this is not the case.

This could be due to the fact that as a single cell organism, yeast does not have the same ability to selectively absorb organic compound that a more complicated organism might have. This would mean that yeast would have a) more waste product to deal with, as a higher number of products it absorb it would be unable to use and/or b) it would have to carry out a higher number of reactions to convert useless products to useful ones. Both these things would undoubtedly increase yeast's metabolic rate and therefore, its concentration of catalase. Also, as a single cell organism, the whole organism would be involved in the investigation, whereas with the other tissues only parts of the organism would be involved. This is important because different parts of different organism would have different metabolic rates depending on its function, therefore the concentration of catalase would vary from area to area.

The differences in the initial rate from dried yeast to fresh yeast is about double, this could be due to the fact that the number of enzymes could be decrease by the drying process. The difference between muscle tissue and liver tissue is also about double this is probably due to the different function of both. It would seem that the muscles were more active than the liver; therefore they would have a higher metabolic rate, which would explain the results. However, this may not necessarily be the case, further research would have to be undertaken to find out the real reasons for these results. The difference between potato and celery is not as marked as the others being only 0.1 out. This is probably due to the differences in how they grow and what their functions are.

To work out the initial rate of reaction the volume produce in the second 30 seconds of the experiment (30 to 60 seconds) was used due to the difficulty involved in recording the volume for the first 30 seconds (0 to 30 seconds), especially in the yeast experiment since the volume of gas produce was at a very high rate.

Conclusion

The most important limitation to this experiment was the difficulty in obtaining an accurate reading on the volume of oxygen produce at a set time, as the stop watch also had to be watched to know the time. This meant that the readings for the volume were sometimes not exactly at the right time. This could have lead to some anomalous results.

Another limitation is the temperature. The experiment was performed at room temperature; this can vary from day to day and therefore might have had an effect on the catalase enzyme. Also, another limitation would be the air tightness of the apparatus. The problem is that there are a number of areas in the apparatus where it cannot be guaranteed to be completely airtight. This would have an effect on the experiment as it would allow some of the oxygen produce to escape, therefore the readings obtain would not be precise, as it does not account for the complete volume of oxygen produce.

The biggest limitation of the experiment was the time constraints. To obtain to sets of results from each type of tissue required 12 experiments to be carried out, this added with the 3 which were discounted due to the problem with the amount of tissues used meant that there was no time to repeat the experiment or to test other types of tissue.

Overall, the investigation does appear to produce some evidence to support my hypothesis that animal and fungi tissues would have a higher concentration of catalyse than plant tissues. However, numerous repeats of these experiments, as well as further experiments with a wider range of tissues, e.g. multi-cellular fungi, at a wider range of times, would clearly be needed before any firm conclusion could be drawn. For instance, the range of tissues could be expanded to include not only multi -cellular fungi, as mention above, but also flowering plant and non-flowering plant tissues, as well as, a wider range of animal tissues, not only from different parts of the animal but also from different animals. For this particular experiment, however, the best improvement would be more repeats at a wider range of time, e.g. every 20 seconds instead of every 3o seconds.