Graph from “Introduction to Advanced Biology” By CJ Clegg, 2000 John Murray
Factors affecting enzyme activity
There are many factors that affect the catalysing action of enzymes. My investigation is directed only at the effect inhibitors have on enzyme molecules. Because of this, all other factors will be kept the same throughout the experiment to avoid introducing other variables. Factors that affect the rate of an enzyme-controlled reaction are:
- Temperature
- pH of Solution
- Substrate concentration
- Enzyme concentration
- Presence of an inhibitor
To maximise the rate of reaction, I will keep my yeast (and therefore the enzymes) at 37 degrees centigrade by means of a water bath. This temperature has been chosen as it is the optimum temperature for the enzymes to operate at. Beyond this point, although the number of collisions with the active site is increased (the kinetic theory,) The temperature causes the enzyme to denature (change shape) and therefore become ineffective
The enzymes used in this experiment
To investigate the affect of inhibitors on enzyme activity, a measurable product must be created as a product of the reaction. This product must be separable from other chemicals in order that its mass or volume can be recorded.
In this investigation, the respiration of yeast was seen as a good solution. Yeast respire, producing CO2 gas. As respiration is an enzyme controlled series of reactions, introducing an inhibitor will affect the rate of respiration, and thus the volume of gas collected. Yeast also respire both aerobically and anaerobically. This is useful as the reaction will not stop if there is no air in the dough which the yeast are suspended in.
Initially mitochondria were to be used, but the process of extraction was deemed too complicated for the limited time available.
Aim
The aim of this experiment is to observe how enzyme inhibitors affect the catalysing properties of enzymes (enzyme action,) using inhibitors for enzymes involved in the respiration of yeast.
Readings of the volume of CO2 gas released (a product of respiration) will be taken using different inhibitors at a set range of inhibitor concentrations. For example inhibitor ‘x’ will be tested at concentrations between 0% and 2% at 0.2% increments. Inhibitor ‘y’ will also be tested at the concentrations specified for ‘x’ and in the same conditions.
Hypothesis and Prediction
I predict that the inhibitor will affect the respiration of yeast by impeding enzymes essential to the process. The greater the concentration of inhibitor, the greater the number of immobilised enzymes, and therefore less CO2 gas will be collected due to respiration slowing down. Eventually, respiration will come to a complete stop, when the inhibitor concentration is so high that all enzymes in the respiratory pathway have been inhibited.
I will use a variety of inhibitors to demonstrate that the effect is due to the inhibition of enzymes rather than a specific chemical reaction between carbon dioxide and the inhibitor. I predict that all my chosen inhibitors will affect the production of gas in a similar manner. By this I mean the volume of gas will be inversely proportional to the inhibitor concentration for all inhibitors.
I have already explained in my background my reasons for choosing yeast suspended in dough for my source of enzymes. I have also explained how heavy metals act as inhibitors. I will be using salts of these metals (which I predict will have the same effect. This is based on chemistry, not biology and so will not be covered in detail here.
Chemical and equipment list
For this investigation, the following chemicals will be required:
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1% Concentration - 20cm3 of solution
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1% Concentration - 20cm3 of solution
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1% Concentration – 20cm3 of solution
The following apparatus is also necessary:
- Swan neck’ glass capillary tubing
- Suitable boiling tube sized bung placed on one end.
- For immersion of capillary tube and measuring cylinder
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For collection of CO2 gas
- With heat proof mat and tripod
- Digital Timer
- Tray suitable for mixing flour and water.
- Rubber gloves
Planned Method
Set up the experiment as shown in the diagram below:
Having assembled the above set up, the yeast, flour, water and inhibitor must be homogenised in the correct proportions. For the size of the boiling tube, the most appropriate amount of flour to make the dough will be 15g, combined with 9 cm3 of distilled water. This forms the bulk of the mixture. To the dough, the yeast is now added. For this amount of dough, 0.3g of yeast are sufficient. The inhibitor solution will also be added at this point. For one run however, no yeast will be added to the dough. This is a control to ensure that it is the enzymes in the yeast are causing the release of carbon dioxide and nothing else. 1 cm3 of inhibitor (of varying concentration) will be added to each dough mixture
The dough must now be thoroughly kneaded (squashed and pulled) to create air pockets, providing oxygen for the yeast to respire aerobically. The finished dough will now be placed into the boiling tube and the caplillary tubing connected. At the other end of the tubing, the tub will be filled with water, as will the measuring cylinder. The tubing will then be placed into the tub as shown in the diagram, and the measuring cylinder (now with no gas in it at all) placed over the end of the tube. The boiling tube will be placed into the water bath to increase the rate of reaction. At this point the timer will be started. At 30 second intervals, the volume of gas will be recorded. The total time spent collecting data will be 10 minutes for each test tube.
For each of the three inhibitors being investigated, the experiment will be repeated four times to minimize experimental error and improve reliability.
Safety precautions
In the course of this investigation, I will be exposed to several different hazards. Firstly, the inhibitors I am using are poisons, which if ingested in large quantities could cause serious illness or death. I will therefore wear gloves at all times, avoid touching my mouth or facial area during the practical, and ensure all surfaces are wiped clean of any spilt chemicals.
I will also be handling fire when using the Bunsen burner. To avoid catching any loose clothing in the flame, I will remove any scarves, coats etc that pose a hazard. I will also turn the Bunsen onto yellow flame when not in use, as this is clearly visible to other people, and is cooler than a blue flame, so less dangerous.
Another hazard is the possible breakage of glassware through overheating, sudden cooling or just dropping a beaker. I will therefore exercise extreme caution when handling glass, and make sure I do not place a hot beaker or test tube into cold water. I will also wear safety goggles throughout to protect against fragments of broken glass, and splashing chemicals.
I have also considered the ethical issues involved in my investigation. I have come to the conclusion that there will be no cruelty or inhumane treatment inflicted on humans or animals throughout the experiments.
Method
In practice, the experiment was set up as detailed in the diagram below:
The planned apparatus was assembled as intended. The following chemicals were present in the following volumes and concentrations:
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500cm3 Distilled water bottle
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20cm3 1% concentration ZnNO3
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20cm3 1% concentration PbNO3
- 15g Dried yeast
- 1kg sifted wheat flour
To begin with, the chemicals were measured out using the 10m3 measuring cylinder and the balance. 9cm3 of distilled water was added to 1cm3 of ZnNO3 / PbNO3 / H2O using the measuring cylinder. 0.3g of yeast was measured out along with 15g of flour. A balance accurate to 0.01g was used to ensure accurate measurement of the mass of the yeast. The dry substances were measured in an evaporation basin, as this was an ideal shape to hold the mass of flour used. It had high sides, stopping flour from spilling out.
The flour and yeast were then placed in a plastic 250cm3 beaker whereupon the water and where appropriate, the inhibitor, were added. The chemicals were mixed using a glass rod. The dough was mixed until any dry mass of flour and yeast had been absorbed into the piece of dough.
The dough was then rolled by hand into a long cylindrical shape, to allow it to pass into the top of a boiling tube without adhering to the sides. This was important as all the dough needed to be at the bottom of the tube, so any expansion of the mixture would be upwards, compressing the air above the tube only, rather than simply filling air pockets below the dough. The dough was pushed to the bottom using a glass rod. The tube was then placed in a 1dm3 beaker filled with water maintained at a temperature of 40 degrees centigrade (a water bath.) The temperature of the water was monitored by means of a thermometer, and heated periodically when necessary using a Bunsen burner.
The boiling tube was connected to a glass capillary tube using a bung. The tube carried gas from the test tube into a tub of water, where the gas would bubble up. This gas was collected using a measuring cylinder (filled with water and placed over the end of the capillary tube.) For the first two minutes, however, the gas was allowed to bubble out without being collected. This short period of time was allotted to allow the dough to heat up to the temperature of the water bath, giving a constant temperature throughout the experiment.
The measuring cylinder was then immersed in water to remove any air trapped inside, and placed over the capillary tube as shown in the diagram. At this point, the stopwatch was activated, and the volume of gas collected in the measuring cylinder recorded at 30-second intervals for 10 minutes. After the results were recorded, the tube was disconnected from the capillary tube to avoid the dough expanding into the pipe. The dough was removed from the boiling tube using a glass rod, the tube then being rinsed in tap water.
The first sets of results were attained using no inhibitor. 15g of flour, 0.3g of yeast and 10cm3 of water were the substances used to create the dough. The second sets of results were arrived at using the same quantities of flour and yeast, 9cm3 of water, and 1cm3 of 1% concentration Zinc nitrate. The third set of results were set up almost identically to the former, but using 1% Lead nitrate in place of the Zinc nitrate.
For the fourth and fifth collection of readings, a different concentration of inhibitor was used. Instead of 1% concentration inhibitor, the chemicals were diluted to 0.5% conc. This was achieved by mixing 5cm3 of Inhibitor with 5cm3 of distilled water in a measuring cylinder. 1cm3 of this was then removed and added to 9cm3 of distilled water using a pipette. Readings were then taken for 0.5% concentration ZnNO3 and PbNO3.
The final group of data was from the control. This was a mixture of flour and water (15g, 10cm3.) The control was to ensure that any gas released in the dough was a result of the metabolic reactions taking place within the yeast and not, say, the expansion of air in the test tube. Therefore, the control was monitored for 10 minutes to see if any gas would be released.
Every concentration was repeated 3 times to ensure reliability of data, and to show up any anomalous results. The control was not repeated due to time constraints.
Analysis
The purpose of this analysis is to identify any trends, patterns or correlations in the results detailed above.
The first trend that can be noticed when analysing the results from the experiment is that over the measured time period, the volume of gas collected by the measuring cylinder increased. When inhibitors were added to the dough, the volume of gas, and the rate of gas collection decreased. This demonstrates that the production of carbon dioxide (a known bi-product of respiration) is directly linked to the rate of respiration. This is significant as it allows the rate of gas collection to be used as a measure of the rate of respiration within the yeast (which was the initial aim of the investigation.)
Looking more closely at part of the data, one can clearly note that the rate at which gas is collected at the beginning of the reaction is slower than toward the end where it speeds up. If we examine data from the experiment with no inhibitor present, we can see that between 30 seconds and 2 minutes, only 7.89% of the total volume of gas had been collected. However, later in the experiment, between 8 minutes and 9 and half minutes, 25.09% of the total gas was collected. This increase in rate of reaction is, however unique to the dough containing no inhibitor.
When Zinc Nitrate (0.5% concentration) was present, the percentage of the total volume of gas collected between 30 seconds and 2.5 minutes was 24%, while between 8 and 9.5 minutes, it was 15.2%. This trend is followed by all the other experiments (excluding the control.)
A possible explanation for why the rate of reaction of the inhibitor-free experiment increased is that it took a few minutes for the yeast cells to re activate from their dried state. Their cytoplasm needed to re-hydrate by osmosis, which took a few moments, during which the reaction proceeded more slowly as the concentrations within the organelles of the yeast changed and moved toward their optimum.
One possible reason for this is the inhibitor itself has an optimum temperature at which it can collide with enzyme molecules and latch on. If it is a reversible inhibitor then it will be removed again, but if the rate of reaction is high enough, the probability another inhibitor molecule will take its place is very high. What may have happened is that at the beginning of the experiments with the inhibitor present, the temperature had not yet reached this optimum for the rate of reaction. As the boiling tube remained in the water bath, however the temperature of the dough will have increased, causing the inhibitor to become more effective and thus affect the overall rate of reaction.
Another possible explanation for the gradual decrease in rate of reaction is the diffusion of the inhibitor molecules into the yeast cells and their mitochondria. To explain, the inhibitor will only function when it is present within the yeast, and within the mitochondria. At the beginning of the experiment when the yeast and inhibitor had only just been mixed together, not many of the inhibitor molecules will have diffused into the yeast; they would still be in the dough mixture, and so not affecting the respiratory chains. As the experiment proceeded however, more and more inhibitor will have diffused into the mitochondria and affected the enzyme controlled reactions central to respiration. This hypothesis seems the more plausible of the two.
The increases and decreases in rate of reaction are very gradual however, which is why the graphs look relatively straight, even though one would expect a more curved graph in such a situation.
In this investigation, inhibitors of varying concentrations were used in the experiments to support the hypothesis that a greater concentration of inhibitor molecules will deactivate a larger number of enzymes and consequently decrease the rate of an enzyme-controlled reaction. From this theory, the higher concentration inhibitor should have produced a lower rate of reaction compared to the lower concentration of inhibitor. To some extent, the results attained support this statement. The total gas collected over a 10-minute period for the dough mixture containing no inhibitor was 9.30cm3 (average.) For a dough mixture containing 0.5% concentration lead nitrate, the total volume of gas collected was 4.67 cm3. The total gas collected over 10 minutes for dough containing 1% concentration Lead Nitrate was a significantly smaller 2.93cm3. This data clearly shows a decrease in the rate of reaction when inhibitor concentration was altered.
However, despite having data supporting the idea of inhibitors decreasing the rate of reaction, some of my readings also conflict with this. Zinc nitrate (0.5% concentration) dough produced 4.17cm3 in 10 minutes while 1% concentration ZnNO3 4.83cm3. This is more gas, not less as was expected. It seems that these results are anomalous as they do not follow the hypothesis laid out at the beginning of the investigation, nor do they repeat the trend of the results attained using lead nitrate as an inhibitor. I am unable to come to a feasible explanation as to why these results should be as they are, other than some sort of error during the experiment. It is possible that air leaked into the measuring cylinder via the seal around the boiling tube and capillary tubing.. This would create incorrect data.
In addition to using varying concentrations of inhibitor, two different inhibitors of enzyme activity were used to ensure that the effects seen were not due to a chemical reaction (between the inhibitor and the released carbon dioxide perhaps.) Of the two, Lead Nitrate and Zinc Nitrate, Lead nitrate seemed to be more effective at inhibiting respiration, as 4.83cm3 of gas was collected when Zinc was present, while only 2.93cm3 was collected when Lead nitrate of the same concentration (1%) was present. Zinc Nitrate did not behave as expected, however (described in previous paragraph.)
A control experiment was also set up in the investigation. This contained dough with no yeast or inhibitor present. The purpose of this was to ensure that the gas collected was a direct consequence of the respiration of yeast and not due to any other factor. If the gas collected was not related to respiration, then the entire investigation would be incorrect, as the basis of measuring the effectiveness of inhibitors is to observe how the enzyme controlled reactions in respiration ate affected. It is therefore necessary to measure a product of respiration only. The results from the control showed that 0.2cm3 of gas were collected. It was expected that no gas whatsoever would be released. This small amount is most probably to do with the air trapped within the boiling tube and capillary tube being heated by the water bath, expanding and being forced out into the measuring cylinder.
