Limitations to the conclusion
However, there are also some limitations to my conclusion due to the extent of my data, which could affect the confidence I can place in it. This relationship of the rate of reaction increasing is not definite as the error bounds showing the range of results overlap, showing that the true rate of reaction theoretically could be the same at pH 5, 6, 7 and 8. However, the box and whisker plots in Figure 2 show that the majority of the data in the boxes (representing 50% of the results) do not overlap. There is a distinct increase between the majority of data between pH 4 and 5 and then again between pH 5 and 7 (although the boxes overlap between pH 6 and 8). This makes the trend less ambiguous and therefore reliable.
There was also a severe limitation on the conclusion due to the incomplete range of pH values used in the investigation. It allowed only showed part of the curve expected from the scientific theory, displaying mostly the acidic pH values and the rate of reaction increasing and flattening, but there was no pH to entirely prove it decreased in a bell- shape curve after pH 8 as the conclusion and scientific theory suggested. More conviction in the conclusion could have been possible if a higher range than pH 8 had been used and hopefully provided evidence that the rate of reaction falls after pH 8. As, there is no data to substantiate this part of the conclusion, it is currently limited. This would have to be further investigated to prove or disprove the conclusion. The large intervals between the pH also limited the data and made what we had less reliable. If decimal pH values were used, for example measuring the rate of reaction at an interval of every pH 0.5 instead of pH 1, then we would have a better understanding of the exact influence of pH on the reaction. This would have been particularly useful around pH 7 and 8, where the temperature peaks and the optimum temperature. It is difficult to judge the real optimum pH from our data as although the highest rate of reaction is at pH, some of that potatoes are started to decrease in the rate of reaction by pH 8. This shows that the optimum pH could be a little before pH 8. In fact, the actual optimum pH for catalase is 7.6 . However, it was impossible for us to find out with the pH intervals used to see if this was the case with our experiment. This shows that the curve is not as precise as it could be and therefore the conclusion is not the most reliable possible. If we did test at a range of pH with smaller intervals it would help make the data more precise and therefore the conclusion more secure.
There is also an evident fair level of scatter shown by the wide error bars. This shows that that the data was not entirely reliable, perhaps due to method or human error, so mistakes could affect the reliability of the data.
Finally, the conclusion is limited to only the enzyme catalase and does not account for the influence pH has on other enzymes, as although many enzymes behave in a similar pattern, pH can have a completely different effect. For example, pepsin which is an enzyme that breaks down protein has a highly acidic optimum pH of 2.
For these reasons we cannot put complete faith in the conclusion.
Scientific Explanation of Conclusion
However, the science explains the current conclusion.
The reason pH effects the rate of enzyme reaction is because of how it can change the shape of the enzyme. As described in the introduction, enzymes are proteins made from long polymer chains of amino acids, which are extremely tightly folded into a particular 3D protein shape called a globular protein. The amino acid chains of which the enzyme is composed of are made of lots of atoms in a particular order held in their shape very tightly together by covalent bonds. The amino acids are then help by ionic bonds in their 3D tertiary shape. The shape is extremely important for the enzyme to work. For the enzyme to break down a molecule, this substrate must fit exactly into the active site, the place where the reaction takes place. The active site is made of chain amino acids with atoms in a particular order to match the substrate. The particular series of the atoms in the amino acid chain at the active site form temporary ionic bonds to the substrate to hold onto it before it is broken down.
The best model of enzyme function is imagining the enzyme active site to be a lock and the substrate to be a key. So, if the shape of the lock changes, then the key will no longer fit. This roughly reflects what happens when the active site changes, as the substrate molecule will no longer fit in it. Therefore, the reaction the enzyme catalyses will not take place. This change of the active site shape is called denaturing of the enzyme.
When we alter the pH we are changing the concentration of the hydrogen ions in a solution. These are what have the effect on the shape of the enzyme. A change in pH can make the charge along the active site can change, stopping the substrate forming ionic bonds with it. When the pH is more acidic and there are more H+ ions, a negative ion on the active site can pick up a positive hydrogen ion. Therefore it becomes neutral and cannot form an ionic bond with the substrate and the enzyme cannot properly work.
This is shown in the two following diagrams:
Therefore, there are fewer reactions between the enzyme and substrate and thus the rate of reaction decreases. The same idea occurs when the solution become more alkaline, the positive ions on the active site can loose their hydrogen ion also becoming neutral and preventing ionic bonds to form to the substrate.
Altering the pH extremely affects the enzyme even more. At very high or low pH’s the ionic bonds with keep the enzyme in its tertiary shape can be affected. This means the enzyme completely looses its shape and the active site will probably be lost completely. When the amount of hydrogen ions does not affect the charge of the active site or ionic bonds enzyme the shape is not affected and the enzyme works best. This is the optimum pH.
The reason that the curve is gradual and then become steeper is because of the different effect the strength of pH has. The slight variation of pH from the optimum which change the charge on the active site is only a gently curve as if there are only a few more or less hydrogen ions only a few of the enzymes will be affected, so the rate of reaction will not be changed that much. This is why curve of rate of reaction on pH is gradual both sides of the optimum pH and there is not much dramatic change in the curve of the graph in Figure 1 between pH 7 and 8. However, once the variation of pH is more considerable the changes affect more enzymes. This is why the curve became steeper as the pH values became more acidic than pH 7. When the pH became so acidic and the whole enzyme’s ionic bonds began to be changed and the enzyme active site denature the graph fell steeply as shown between pH 5 and 4. When all the enzymes were completely denatured there would be no reaction at all.
Evaluation
Evaluation of procedures
The experiment had a simple method, which was easy to repeat in the same way again. It was also very safe to conduct, if you followed the explicit safety precautions wearing safety goggles and lab coats and being highly careful with the highly oxidising hydrogen peroxide and dangerous apparatus such as knives or broken glass if a test or manometer tube was broken.
It was completed in a reasonable amount of time and it gave a wide set of data that could be studied and analysed.
However, it the data was not as reliable as it could be. The range of rate of reaction for each pH was huge. For example at pH 8 the results ranged from 37 to 134 mm/20s. This meant that there was a large degree of overlap between the error bars representing the range of values, and even the boxes in Figure 2 showing the majority of the data in the inter quartile range, at the different pH. This means we cannot be certain in the conclusion as the true value could fall anywhere in the range. It shows that the data collected was not particularly accurate.
The fact that the results were not completely accurate could be due to the method as there were several experimental flaws in the investigation and there are a number of improvements that could be made to improve them and therefore get a more accurate set of data to make a reliable conclusion from. These were the main problems and a suggested improvement:
1. Issues with the apparatus/techniques
Measuring the rate of reaction by seeing how far the oxygen produced evolved the manometer fluid was an inaccurate method. Firstly, it was essential to hold the monometer tube completely vertically to measure the movement precisely. However, it was easy to slightly tilt it and once the oxygen had started moving the fluid you could not check from the marker if it was help in the correct way. This meant that the when we measured the distance the oxygen had evolved it could be a few millimetres wrong.
This could have been prevented if the manometer tube was help in place by a clamp so it was always exactly the same angle. Holding at the same angle would stop the human error of moving it slightly and changing the results.
Moreover, the measurements also required the use of rubber tube folded over as a seal to prevent any oxygen escaping. However, there was still a great chance of oxygen leaking out as you could not hold the tube completely sealed and the pressure holding it down could change as fingers got tired. This meant that the reading of the oxygen evolvement was not as accurate as it could have been. Using a three way clip might have prevented this because you can turn the tap rather than having to personally hold it, so there is less opportunity for human error and the oxygen escaping.
A better alternative might also be not to use the manometer tube at all but to measure precisely how much gas was produced in a certain amount of time by volume metric. For example allowing the oxygen created to displace water in graduated cylinder held underwater, so you could read exactly how much oxygen was produced in a certain amount of time. This would give an indication of the exact volume of gas created from the enzyme reaction in a certain time, rather than just comparing how far the oxygen created moved the manometer fluid which showed the relative rate of reaction. It would be a more precise measure with quantity of product to show the effect pH had on the enzyme.
Some of the measuring apparatus was not very accurate. The syringes often collected bubbles varying the volume of hydrogen peroxide that was put in each test tube. This could have affected the results as different volumes of hydrogen peroxide would have changed the rate of reaction of the enzyme. If we wanted to be more precise we could have used a burette to measure out the hydrogen peroxide as a burette has a higher degree of accuracy.
Timing was also a significant issue. Every different measurement there was capacity for there to be a different time delay before starting the stop watch. It was impossible to start it at exactly the same point with every single measurement. Therefore, the values are likely to be a few mm/20s from their true value. Perhaps using an assistant just for timing would have overcome the timing issue as they could begin timing quicker without the hindrance of having to switch apparatus before starting the stop watch. They would also know when exactly the started and stopped the reaction on the other measurements, so could repeat this every measurement. This would have made the times more accurate to their actual rate of reaction. However, it is still not completely accurate as there would still be opportunity for time delay.
2. Not controlling variables
It is important to control the variables of the experiment otherwise they can also affect the results and this makes the results not only about the effect of pH. However, the method did not manage to control all the possible variables. Temperature also has a significant effect on the rate of enzyme reactions, so it should have been controlled as having different temperatures would have given very different readings. Over our experiment the temperature of the classroom could have changed by a couple of degrees centigrade as the people warmed the air, or it could have cooled if somebody opened a window or door and allowed a draft. Different areas of the classroom would have also been different temperatures depending on if they were near the radiator or door etc. This could have changed the rate of reaction of the enzyme and our results. Therefore, to control the variable we should have placed the test tube of hydrogen peroxide and the boiling tube of potato disks and pH buffer in a thermostatically controlled water bath, set at a pre-decided temperature, and let them acclimatise before adding the hydrogen peroxide. This would make sure that the enzyme reacting at the same temperature every time, so different temperatures could not affect the reaction and the results we got would be solely due to the affect of changing the pH.
The concentration of the substrate (hydrogen peroxide) could also vary as over time as it got broken down by the catalase. So, when the concentration got less the reaction would have got slower. If we took a different amount of time before actually timing the reaction, the concentration would have changed and therefore the results would be different to how they would have been if they were exactly the same concentration. This would cause some variation within the data it is not as accurate so looking at it is not reliable. Therefore, we should start timing immediately after the hydrogen peroxide has been added so the concentration will be the same with every measurement and could not vary the results.
Another variable was the amount of time that the potatoes were left in the pH buffer solution before the addition of the hydrogen peroxide. This could vary how much of the pH the potato could soak up and therefore how much the catalase in the potato was affected by pH. We should have left the potato discs in the pH for a given time so that they would absorb the exactly the same amount of pH each time. This would be better than reacting with the hydrogen peroxide immediately as we did as it would give more chance for the pH to be absorbed so the catalase was affected more. This should give a greater difference between our results so we could see the trend would be clearer and therefore it would be more reliable to base a conclusion on.
3. Limit of the Range
As I have already described in the limitations of the conclusion section, the range of pH values was not extensive enough to get a very good set of results. The current range was too small to be able to see the affect of more alkaline pH on catalase. This meant we could not see the full extent of the effect pH has on catalase so part of our experiment is incomplete. We would have to conduct the experiment in the same way again at more alkaline pH values to see what happens and to make our conclusion more secure by having appropriate data to back it up. It also did not use small enough intervals to examine in more detail the exact effect around pH 7 and 8 and to ascertain the exact optimum pH for catalase.
Reliability of Evidence
Due to these issues with the way we conducted our experiment, it is clear that they will affect our data and there will be some variation of the results and they are not as accurate as they could be. This is why there was a degree of scatter shown by the large error bounds. Even some of the inter quartile ranges were quite large, particularly at pH 6 and 8. This shows that the results were all generally over a very large range at these pH values not just a few of them, highlighting the degree of scatter.
The issues could also account for the three anomalous results of potato 3 at pH 4. However, these results are so out of sync from the rest of the results and do not follow the trend of all the other results to such an extent that it is possible that they are not just the fault of the apparatus of human error and beyond the range of experimental data. Perhaps that somehow the pH buffer liquid could have got confused or mislabelled.
Another weakness in the data that affects the overall reliability of the evidence is the severe lack of data for pH 6. Due to an error in the labelling of the pH buffer bottles, unfortunately only two groups were able to collect data for this pH. Since we only have two sets of data from only two potatoes it limits the accuracy of the mean as there is less data to work out the closest mean to the true value it is less likely to as accurate. This is why the inter quartile range for pH 6 is so large and unlike the rest of the values. It makes the conclusion less reliable as we do not have a particularly accurate basis to judge the effect of pH 6 on the enzyme. This could affect the general trend of the data. Further data collection at this pH would make the data more reliable to base the conclusion on.
On the other hand, some aspects of the data and investigation make it quite reliable. We used a range of eight different potatoes and although this caused a wide variation is important to use a range of different potatoes as it represents the wide variability in different biological material. All different potatoes are different depending on their age, health, amount of catalase and it was important to represent that in our experiment. The fact that all eight of the different potatoes shows the same trend makes the experiment more conclusive as it shows that a wide array of varying biological matter all behave the same way showing that pH has the same effect on all potatoes.
Using eight potatoes and repeating the rate of reaction of each three times at the different pH values (apart from pH 6) meant we had a wide set of data to show clearly a repeated trend and clearly observe any outliers. The more results taken the more accurate the mean was.
This fact that all biological material varies partly explains the generally large, overlapping error bars (however they are also likely to be slightly due to the faults in the method and human error). For example, potato five was overall the pH values one of the slowest. This could be because it is old or sick or simply that it did not have that much catalase. On the other hand potato 3 had an extremely fast rate of reaction compared with the other potatoes. Its fastest rate of reaction was 134mm/20s. This shows that this potato has a high amount of catalase and is in excellent health. This variation is only normal and if you look at the repeats of the individual potato results the repeats are all reasonably close. For example the repeats of potato 6 only varied at a maximum of 6mm/20s at one pH. This shows that although there were faults in the experiment overall the similar repeats show that there was not too much variation each time so the groups worked with quite a good degree of precision and therefore the results are reliable.
The box and whisker plot graph also shows that the results are reasonably reliable as the inter quartile range (shown by the box) are generally quite small, particularly at pH 4, 5 and 7. This shows that not only was the trend the same for every potato but also that the majority of the data was close showing that even the different potatoes were similarly affected by pH.
Therefore, although there were anomalies, missing data, and some scatter the evidence is on the whole reasonably reliable. Although there are some aspects that could be improved in the procedures to eliminate scatter and outliers and more evidence could be collected at pH 6, which would make the evidence even more reliable.
Reliability of Conclusion
As the general reliability of the evidence because of the close repeat measurements and the same trend throughout the whole data, I would say that my conclusion, “pH affects the enzyme catalase by increasing the rate of reaction of breaking down hydrogen peroxide until around the optimum of pH 8, before it begins to decline” is reasonably accurate and reliable itself.
I am reasonably confident due to the clear upward trend in the data that the increase pH definitely makes the enzyme break down hydrogen peroxide more effectively until around the point of pH 8. Moreover, the data of all eight potatoes showed this trend which means we can put more faith in it.
However, I do not have the same level of confidence that it declines after that point as I do not have a complete range of pH values more alkaline than pH 8 to prove it and only three of the potatoes tested began to decrease by pH 8. Therefore, I cannot be certain even if the scientific theory and some of the data show it.
There are weaknesses in the techniques, as discussed in the ‘Evaluation of Procedures’, which would have affected the accuracy of the data, which limits the faith I can place in the conclusion. This included the lack of precision of some of the apparatus, such as the oxygen escaping from the tube you had to hold down and, also the lack of control of variables. The range was not adequate to see the full effect of the whole spectrum of pH, both acid and alkaline. This is why although I am only reasonably confident I cannot place full confidence in the conclusion.
However, we could have more confidence if we amended these problems by:
- Using the improvements in apparatus and techniques described in the ‘Evaluation of Procedures’ to make the data more accurate and therefore more reliable.
- Extending the range to use more alkaline pH values, such as pH 9, 10, 11, 12 to provide more evidence for the second aspect of the conclusion. It would also help to continue testing more acidic pH values to see when the enzyme was denatured to such an extent that it could not react at all with the hydrogen peroxide. This would display the full effect of pH on catalase for us to base the best conclusion on.
- Including pH values at smaller intervals to ascertain the optimum pH more accurately.
- Taking further data at each pH 6, where there was a lack of it, as to get a better picture of what the effect of this pH is
These ideas would make the data be more accurate and complete, and therefore make the conclusion more reliable and secure.
Source: ‘Twenty First Century Science GCSE Biology’ published by OCR (page 101)
Source: ‘Twenty First Century Science GCSE Biology’ published by OCR (page 101)
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