We carried out a preliminary experiment to see whether our original plan worked. It highlighted a few problems such as the amount of hydrogen peroxide we used. We discovered that if we used too much then the reaction would be over too quickly so we had to reduce the amount of H2O2 we used to 5ml. We also realised that 2g of celery was an adequate amount to be used. As well as the preliminary experiment highlighting some errors with our method it also enabled us to see whether all the equipment worked well with each other providing the most accurate results possible. The following lists the factors that shall remain as constants;
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We will keep the pH the same by using the same concentration of H2O2.
- We will use the same amount of the enzyme catalayse by using the same amount of celery.
- We will keep the temperature range and time reaction the same throughout the investigation.
As a control we will also have two experiments where there is only one substance in the conical flask so one experiment will be carried out solely with H2O2 and the other with only the celery paste containing the enzyme catalayse. Evidently, no reaction will take place and so from this we can prove that it is the adding of the catalayse and H2O2 together that actually causes the production of oxygen, which is what we are testing.
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Fair Test;
To keep the experiment a fair test we can ensure that we can obtain the most accurate results as possible. It will also allow us to ensure that each experiment will be carried out in exactly the same way so that in each of the three experiments, the results are as similar as possible;
- We will use the same batch of celery from which the catalayse will be extracted at the same time. This ensures that the catalayse we use is of the same concentration throughout the whole experiment.
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We will use the same amount of celery by always making sure we have exactly two grams of the mixture before we place it into the H2O2. We will also use the same amount of water in this mixture (about 2ml).
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We will use the same amount of H202 in each experiment
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We will use two controls. These will be one experiment with only celery and one experiment with only H202. This allows us to prove that the reaction cannot take place without actually adding the two substances together and that by themselves there is no reaction.
- I will keep the variable the same along with its range and intervals. (Temperature, 0-70°C with 10°C intervals)
- I will keep the constants the same throughout the experiment.
- I will use the same equipment for every step within each experiment.
- I will keep the same one-minute interval for the reaction to take place the same in each experiment.
- We will give time for the heat to equilibrate between the water bath and the conical flask containing the hydrogen peroxide.
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We will use the same concentration of H2O2 for every experiment.
Safety;
- We will keep the alkali Hydrogen Peroxide away from skin as it can blister the skin dependant upon its concentration.
- Safety spectacles must be worn through out the experiment as if the Hydrogen Peroxide splashes into your eyes it could cause temporary blindness.
- We must keep the Bunsen burner on safety flame when it is not being used so that the flame can clearly be seen to prevent somebody from accidentally being burnt on it.
- We must be careful when handling the heated water to prevent the risk of being scolded.
- We must keep an eye on the chemicals that have already been poured into the beakers to ensure that we don’t get the water mixed up with the Hydrogen Peroxide, which is also a clear and colourless liquid.
Accuracy;
- We will use a small inverted cylinder in the ice cream tub to obtain a higher degree of accuracy.
- We will follow the statements in the fair test as closely as possible.
- Repeat each experiment three times, from which we can obtain an average.
- Make sure that all are equipment has no traces of chemicals left in it from previous use.
- We will use separate pippets for each chemical. This will also apply to other equipment, which will come into contact with either the catalayse or the Hydrogen Peroxide.
Prediction;
Due to a preliminary experiment, I have a fair idea on how enzymes will be affected due to temperature. I believe that at low temperatures the reaction will be quite slow. Although as the temperature increases so will the rate of reaction. I believe that the rate of reaction will be optimum between 30-40°C as this is what our preliminary experiment displayed. Although if the temperature continues to increase further the catalayse will denature and so therefore seize to react. The following displays the pattern that I expect the enzyme catalayse will follow:
The previous diagram illustrates the common enzyme pattern I would expect the catalayse to show when reacting while under different temperatures. From the start it clearly shows the rate happening quite slowly but then rapidly growing faster producing a steep climbing curve. This then begins to level off until the rate reaches its optimum temperature. The rate then begins to decline quite steadily, although the curve is not as steep as before, it then begins to level off at a slower rate. So, based upon this simple pattern I predict that the catalayse would be only beginning to react between 0-10°C, although very slowly as the temperatures are quite cold. The rate of reaction will now be beginning to increase slowly between 10-20°C but then begin to react faster between 20-30°C. I think that the optimum temperature would be between 30-40°C (which is known as the steady state phase). I think this because within our bodies we have many different enzymes which all work at 37°C, as this is the temperature of the inner body. Beyond the optimum temperature I predict that the rate will begin to decline, this will be between 50-60°C. beyond 60°C I predict that the enzymes will denature, as the temperature will be too hot. The following uses the particle theory along with the active site and substrate theory to explain clearly why I think the catalayse would react in this way relative to how the temperature increases.
Scientific Explanation;
Active Site and Substrate Diagram;
Biological catalysts speed up all reactions, which take place within our bodies and in all other organisms. These biological catalysts are made of proteins and are called enzymes. Enzymes are specific because each different reaction needs a different type of enzyme. This is what the above diagram shows. It illustrates how the active site of catalayse is specific to the substrate Hydrogen peroxide that will then brake down into two products; these are water and oxygen. This is the reaction that the enzymes participate in. At low temperatures the enzymes have less energy and so are less active. This means collisions between the catalayse and the H2O2 are lesser along with the reaction rate. Although as the temperature increases towards 50°C, reactions are faster and stronger causing the active site to deform, and no longer be compatible with the H2O2. This causes the enzyme to denature and no longer react. The following diagram illustrates this
Results:
When we carried out our experiment everything went to plan although a few anomalous results cropped up, but this was expected which is why we repeated each experiment three times to achieve a more accurate and common result. These anomalies are highlighted in the results table and are explained in the evaluation.
As we added the H2O2 to the celery containing the catalayse it was clear to see the reaction-taking place. As soon as the two substances came into contact the reaction began to take place and it was easy to see the oxygen bubbles being given off which happened at a rapid rate, which meant that each experiment did not take long; it was resetting the experiment each time that was most time consuming. The following are the results.
*x Anomalous results.
Analysis/Conclusion;
The following graph displays the average of the three results for each experiment and the line of best fit. The graph is also compared to the expected pattern and shows clearly the relationship between the two, which shows us that the results on the whole proved to be successful:
From the above graph, according to the line of best fit, I can say that the results on the whole match what I predicted in my prediction. Between 0-10°C the line matches what I predicted in my prediction where I said that I think that the reaction would be beginning to happen although very slowly. Between 10-20°C the graph shows the reaction rate beginning to happen faster although still quite slowly. Between 20-30°C the graph shows the reaction rate increasing at a rapid rate. As predicted in my prediction the graph also shows the optimum temperature between 30-40°C. I said that the optimum temperature would be at about 37°C rather than in the early thirties and this proved to be correct as the line of best-fit peaks nearer towards the point of 40°C. In my prediction I predicted that beyond 50°C the reaction rate would begin to decline, which proves to be correct according to the graph. In my prediction I said that the catalayse would begin to denature beyond 60°C. The graph does not clearly show this but it if there were no anomalous results at this temperature range (which there were) the line would be clearer and it would be easier to see the line levelling of as the enzymes begin to denature.
On the whole, the results support my original prediction because the line of best fit on the graph matches the graph drawn in my prediction. So therefore I can conclude that at low temperatures there was a slow rate of reaction and at high temperatures there was a high rate of reaction. So the rate of reaction is directly proportional to temperature. So at a lesser temperature there is less energy being transferred into the solution. This means that the particles of H2O2 and the enzyme molecules are moving slower and so there are less collisions between the enzymes and the H2O2 resulting in less reactions, producing less oxygen and water. This is evident in my graph whereby between 0-20ºC there was only a maximum of 11mm3 of O2 being given off. Although as the temperature increased the enzymes gained more energy and started having more collisions resulting in more reactions producing more products. This is evident between 20-30ºC whereby the rate of reaction had increased rapidly and there was a maximum of 23.5mm3 of O2 being given off. At the optimum temperature (which is shown at about 40°C on the graph) the enzymes were working at the fastest they were capable of, we know this because the rate of reaction begins to decline from here, showing us that the temperature is becoming too high. This supports what I said in my prediction whereby I said that I believe the optimum temperature would be around 37ºC because this is the temperature within our bodies that are full of various enzymes. The rate of enzymatic activity does not increase beyond its optimum temperature. This is because enzymes are living organisms so when the temperature begins to get too high, instead of producing a faster rate of reaction the enzyme begins to suffer problems, and decrease in productivity. The energy from the heat becomes to great and although the collisions are coming faster and faster they are proving to be useless because as the hydrogen peroxide molecules collide with the catalayse they cause it’s structure to deform; this is because the collusions are becoming too strong. As a result of this the active site of the enzyme deforms due to the impact thus proving it to be useless. This is known as the enzyme becoming denatured and means that the H2O2 molecules no longer fit into the active site and so no reaction takes place. As the temperature continues to rise, more and more enzymes become denatured until eventually no reactions take place and the enzymes are completely denatured. This is evident from 40ºC onwards.
Evaluation
The following is a duplicate of the results table:
* Anomalies
The table shows 5 anomalies.
Anomaly number 1 may have cropped up because these were our first set of results and we were still having problems with the apparatus as we had only just started the experiment and so there were a few teething problems.
Anomaly number 2 cropped up, as it is an average and reflects anomaly 1.
Anomaly number 3 may have cropped up because competitive inhibitors may have been present within the solution. These compete for the active site so that the substrate H2O2 cannot be broken down as the competitive inhibitor has stolen the active site within the enzyme. The competitive inhibitor can only steal the active site dependant upon the relative concentrations of the inhibitor and the substrate. We could have prevented the competitive inhibitor from working by increasing the concentration of the substrate, which in this case would be the H2O2. Although we could not carry this out as it would go against my fair test points whereby I said that I would use the same concentration of H2O2 for each experiment. The following diagram illustrates how a competitive inhibitor works:
Anomaly number 4 may have occurred due to non-competitive inhibitors. These reduce enzyme activity, which may explain why this reading was so low. They do this by distorting the enzyme conformation by binding to some other part of the enzyme other than the active site. Any factor, which alters the conformation of the enzyme, will alter the shape of the active site. This means that the substrate hydrogen peroxide will now no longer fit into the active site, which has now been distorted. The following diagram illustrates this:
Anomaly number 5 may have occurred due to a lack of activators. Sometimes activators are necessary to complete the structural relationship between the active site and the substrate. What may have occurred in this instance is that there may have been a lack of them so that some of the enzymes will not compatible with the substrate, therefore affecting the rate of reaction.
Another point, which may have caused anomaly number 5, is that it was the last experiment we carried out. By this time the batch of celery would have begun to decompose because the experiment took place over a period of about two weeks. This would have affected the rate of reaction because it would have meant that the enzymes within the celery would have started to die as the decomposition takes place.
Are results were as accurate as they could have, although some problems did occur. This is evident from the anomalous results that cropped up although nothing could have been done about these unless we repeated them because if they were to do with competitive or non-competitive inhibitors we could have done nothing about them. Are results were not accurate enough to make a conclusion from. We could have improved the reliability of this experiment by improving the method greatly. This was because after every experiment was over we had to reset it. This meant that all the equipment had to be disconnected and moved out of place. This was a tedious and very slow way of doing the experiment. I feel the range of readings was sufficient for this experiment. This is because our range, 0-70°C, seemed to contain all the major enzyme activity and that below 0°C or above 70°C the enzymes would just have still been denatured and shown nothing important on the graph. We could have changed the size of our intervals within this range. This was because when we use the celery we cannot be sure how much catalayse is within it.
When comparing the first set of results with the others it is clear to see that there is a bit of variation between them, our only differences reached up to a 4°C gap between them, but nothing significant other than the five major anomalous results. We used intervals of 10°C whereas if we had used 5°C intervals we would have had more results from which we would have had more plots on the graph. From these plots on the graph it would have enabled us to sketch and estimate a line of best fit, which would be more accurate and similar to the enzyme pattern.
Our method could have been improved for accuracy of readings by getting better equipment such as measuring cylinders that go to a greater degree of accuracy. We could also have used pure catalayse if it is possible. Another point that could have improved our method is that when we heat the hydrogen peroxide to the desired temperature within the conical flask it defies the point of the experiment. This was to find out the effect of temperature upon the enzyme catalayse. So maybe instead of just heating the hydrogen peroxide we should have heated the catalayse as well, this may have given us more accurate results. This is because when we add the heated hydrogen peroxide to the unheated catalayse the reaction takes place almost immediately so the enzyme may not be under the full effect of the H2O2 temperature.
I could not have obtained additional evidence from the information I had access to.
After using this graph in my prediction and seeing that it proved to be accurate I can say that this graph is correct and should apply to most enzymes. So I could say that this could be a law, which explains the common pattern of an enzyme under the effect of temperature:
Although this law only applies to one of the two variables which I listed in my planning on page 3. So to extend the enquiry I could look at the other factor, which was to experiment the effect of pH or the effect of substrate upon, enzymes and then I could also apply these experiments to other enzymes within other plants or even animals.
By rajiv pudaruth