Variables affecting the rate of enzyme reaction
PH level is a factor because enzymes will only function well at an optimum pH value; this is normally 7 because enzymes are proteins which are damaged by very acidic or very alkaline conditions. If the pH level changes then the enzymes become denatured and do not function properly.
Temperature is a variable in the experiment because of the nature of enzymes. Enzymes all work best at an optimum temperature, which is usually body temperature at 37C. If the temperature that the enzyme has to work at gets too high, normally 40C it will start to become denatured and their active site becomes permanently distorted which means that the lock and key reaction with the substrate no longer works. Below a certain temperature the enzymes will not function properly because they do not have enough kinetic energy to collide successfully with the substrates. This variable involves the collision theory, which says that higher temperature particles have more energy. This energy (kinetic) enables the particles to move around at a faster speed. If the particles are moving around faster, then they are more likely to collide frequently.
Enzyme concentration is a factor because when the enzyme is highly concentrated there will be more enzyme molecules, and therefore an enzyme molecule will hit a substrate molecule more frequently and cause a reaction. When the enzyme is less concentrated it is less likely that the enzyme molecules will hit a substrate, as frequently and therefore there will be fewer reactions.
Substrate concentration is a factor for similar reasons as above. Hydrogen peroxide is the substrate in this reaction, if it is highly concentrated more reactions will occur producing more water and oxygen, whereas if the substrate was less highly concentrated less reactions would occur.
Prediction:
It is predicted that if the substrate concentration is increased then the rate of catalyse activity will also increase because there will be more molecules, resulting in more chemical reactions caused by successful collisions between the active site of the enzyme (catalase) and the substrate (hydrogen peroxide). I would expect the highest concentration of Hydrogen Peroxide to react quickest because it contains the most molecules. With more of these molecules inside the solution, it is more likely that a collision will take place; molecules must collide in order to react. This means that a reaction is more likely to take place, in a shorter time, making the rate of reaction quicker. The more reactions that take place increases the volume of oxygen produced in the shortest amount of time. The increase in reactions cause there to be more carbon dioxide given off which proves that the rate of decomposition will have increased.
From my scientific knowledge and with the aid of textbooks, Encarta and the Internet it is also possible to draw a predicted graph.
The graph shows that provided there are a sufficient amount of enzymes to react with the substrate molecules, the rate of reaction will be greater as the substrate concentration increases. The point at the top of the graph where the line levels out is where there are not a sufficient number of enzymes to react with the substrate molecules so the volume of O2 produced has reached it’s optimum point for the number of enzymes available. The line at the bottom levels out because there are no substrate molecules for the enzymes to react with.
Preliminary Work:
It is necessary to do some preliminary work to find out what apparatus needs to be used and what method and measurements are most efficient and accurate. I decided to use a burette, which will be filled with distilled water and clamped in a water bath with a delivery tube, which will lead off from the chronicle flask and release the O2 into the burette. This is the clearest and most accurate way of recording the results using the equipment available, as it is easy to read the volume off the side of the burette. The other option is to count the bubbles released into a measuring cylinder however this is not very accurate as bubbles can vary in size and be easily missed.
For the preliminary work the apparatus was set up as shown
In the preliminary work a range of concentrations were tested to ensure that when the experiment was carried out and the results recorded the range of concentrations would give good accurate results, with a range wide enough to make a fair test and give a correct conclusion.
The volume of celery and water solution was tested because there needed to be a balance between the mass of celery and the volume of water used because the solution had to be the right consistency so that it could be measured accurately in a measuring cylinder and water was also needed to increase surface area and make it possible to break down the celery in a food processor. This was necessary to make the enzyme (catalase) accessible for the reaction. The celery solution contained 100grams of celery and 100ml of water and was blended for 15secs.
The quantity of celery solution and Hydrogen Peroxide used were also important because if too much of either substance was used it would be very hard to record accurate results because in a very small amount of time a large volume of oxygen would be produced. This means it would make sense to use smaller quantities and achieve better quality results.
The concentration of the substrate was tested in the preliminary results to insure the results were as accurate and reliable as possible.
The first test I carried out used 50cm3 of celery solution and 10cm³ hydrogen peroxide solution with the concentration being pure hydrogen peroxide. I put the celery solution in a chronicle flask and added the hydrogen peroxide solution. I put the bung and delivery tube over the flask and left it for 1 minute. However there was too much celery solution and the substrate concentration was too great because the reaction took place much to quickly to record accurate results with the equipment I had. I then tried the experiment using 10cm3 of celery solution and Hydrogen Peroxide with the concentration of 5cm3 of distilled water and 5cm3 of Hydrogen Peroxide. This test worked better and I could record the volume of oxygen given off in 1 minute using the equipment available. I then decided on several other concentration values and tested them to ensure they worked.
I tested the following:
Celery solution= 10cm3
Hydrogen Peroxide solution = 10cm³
Concentrations = 5/5, 4/6, 3/7, 2/8, 1/9
The celery solution and hydrogen peroxide solution was left to react for 1 minute and then recorded the volume of oxygen produced. These values worked efficiently and gave a good range of results. It was also decided to use a burette instead of a measuring cylinder as burettes give more accurate readings.
Range, Fair Test, Accuracy, Safety
The preliminary enquiry helped to discover some valuable points, which will need to be used in order for the final method to be as accurate as possible. To make the experiment a fair test the variables all have to be exactly the same except for the concentration of the hydrogen peroxide solution, this will ensure that the substrate concentration will be the only factor affecting the rate of enzyme reaction.
- The volume of celery solution will not be adjusted, as the same solution will be used for each different substrate concentration tested. The solution contains 50% celery and 50% water.
- The volume of hydrogen peroxide solution will also remain the same but the concentration will vary.
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The volume of celery solution used for the different concentrations will be 10cm3.
- The pH level will not be altered because the same solution will be used.
- None of the solutions will be stirred and they will all use the same apparatus.
- The equipment should be kept the same to ensure all results are taken without any advantages or disadvantages.
- The celery will be kept covered as much as possible and will only make contact with the Hydrogen Peroxide when the stopwatch is started.
- The different concentrations will all be timed for 1 minute.
To insure the test is as reliable as possible the experiment will be repeated three times for each of the varying concentrations.
To make sure the experiment is carried out safely and fairly safety goggles will be worn, as Hydrogen Peroxide can be dangerous if it gets into your eyes. All laboratory surfaces will be cleared and other lab rules must be followed. When the celery is measured out on the scales some polythene will be placed under it so that none of the celery is absorbed into the scales. All measurements of celery will be made to 2d.p. This will increase accuracy because the minimum and maximum it can weigh will be 0.995g and 1.005g. If it was measured to the nearest gram, the measurements could be from 0.5g to 1.5g, which would be totally inaccurate and would make the experiment unfair as massess could vary.
METHOD:
To carry out the experiment the apparatus will be set out as shown
By taking three repeat readings of the experiment it will mean that in my results I can use the average number, which will help make my results more accurate. It also increases the efficiency of my results.
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100cm3 of celery was blended for 15seconds with 100cm3 of distilled water
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10cm3 of the celery solution was measured into a chronicle flask.
- 10cm³ hydrogen peroxide concentration 5/5 was added.
- The bung was firmly pushed on top.
- The delivery tube led to the burette, already filled with distilled water.
- The reaction was timed for 1 minute.
- The volume of gas produced was recorded.
- The reacted celery solution and hydrogen peroxide were safely disposed of.
The experiment was repeated 3 times for each of the following concentrations 5/5, 4/6, 3/7, 2/8, 1/9.
Obtaining evidence
Were any changes made to the experiment?
There were no changes made to the method during the experiment. The volume of hydrogen peroxide solution remained at 10cm³, as did the volume of celery solution. This made it possible to calculate the volume of oxygen produced accurately. It was decided that the length of time left for the enzyme to react with the catalase would also remain at 1minute. The substrate concentration in the tables shows the volume of hydrogen peroxide/the volume of water
Results
The experiment was repeated three times using the same five concentrations in order to obtain more reliable results. From the results the average of each concentration was found so a more accurate graph could be drawn.
These are the figures used for plotting the graph:
I also had to work out the rate at which the enzymes and catalysts react. I did this by dividing the amount of gas given off by the time.
These were the results.
Analysis of Results
From this experiment I have discovered that as the substrate concentration increased so did the rate of reaction. This is because when the substrate concentration was increased there were then more molecules available to collide and react with the active sites of the enzymes. This shows that my prediction was correct. That the highest concentration would produce the highest volume of O2.
The two graphs both resemble the predicted graph as far as possible and all the points are very close to the line of best fit, which helps to show the accuracy of my results. The graphs show a steady decline in the reaction rate as the substrate concentration decreased which is shown clearly in the trend line of the graphs.
To display the information discovered, clearly on the graphs I used the results obtained from my experiment but with the following equations transformed them into data, which could be clearly understood on a graph.
Firstly I found the average for each concentration using my repeat readings.
Repeat 1 + Repeat 2 + Repeat 3 – 3 = Average amount of O2 produced
I used this equation to form information that I could then use to draw a reliable graph for the results I had obtained and it shows the average amount of O2 produced for each substrate concentration.
I then used the following equation to find the rate of reaction for each substrate concentration.
Average amount of O2 produced
—————————————-------- = Rate of reaction
Time taken (seconds)
Example
23.5
—— = 0.39cm3
60
Another observation, which would have made the graph more reliable, would have been to have experimented with more concentrations and see how they related to the results collected from this investigation. If I had, had more time I would have used a wider range of substrate concentrations which would have hopefully supported the data already obtained.
There were no anomalous results in this experiment. All the results are similar and produce a clear and accurate graph as all the points are very close to the line of best fit. Repeating the experiment helped to prove the reliability of the experiment and the similarity of the results shows the accuracy in the experiment.
In the method I tried to reduce inaccuracies by using the same person to start and stop the stop clock, which limited the inaccuracies in reaction time, which can be a problem. I used the same equipment and tried to be as accurate as possible with my measurements.
The results are supported by my prediction and scientific knowledge of the collision theory and enzyme activity. The collision theory states that for an effective reaction the molecules, in this case enzyme and substrate must possess enough energy to collide and react together efficiently. This is obviously present as the results show that effective reactions have taken place between the enzyme, catalase and the substrate Hydrogen peroxide. My knowledge of enzyme activity helped to show what conditions were needed for successful reactions. From the results and graphs I conclude that my prediction was correct. The results were accurate enough to prove this and provide a clear graph displaying the break down of hydrogen peroxide when using the enzyme, catalase.
Evaluation
In the analysis I noticed that there were a few anomalous results, which are in some way connected to the method. In the evaluation I will investigate these as well as finding out possible improvements.
What were the inaccuracies of the experiment?
The results collected were fairly reliable, meaning that they were mostly expected. However, there were some inaccuracies in the experiment due to human error but no anomalous results. However the accuracy of the results could have been improved further by repeating the experiment again. The different substrate concentrations were all kept under the same conditions, as were the enzymes. I also insured the Hydrogen peroxide and celery came from the same place so the results could be as reliable as possible. However it could be that the volume of enzymes in 10cm3 of celery solution varied slightly, which could have caused slight inaccuracies. Any slight differences in these results could also be due to human reaction time. I tried to avoid this by having the same person monitoring the time throughout the whole experiment but it could cause very slight inaccuracies. Overall I think the results obtained and the graph show a clear evidence for the experiment and effective and reliable conclusions can be drawn from this investigation.
How would I improve my method?
My first alteration to the experiment is that I would repeat the experiment again for the two concentrations that had the widest result range. Therefore the results when finding the average volume of O2 produced would be more valid.
I would also repeat the same experiment with a larger range of concentrations. Thus would mean that the graph drawn could be more complete. It is clear from the line of best fit and the trend of the results, I would only have been able to test 6cm3 hydrogen peroxide/ 4cm3 water and 0cm3 hydrogen peroxide/ 10cm3 water. If I had, had time I would have liked to have tested these values.
In this experiment 5 substrate concentrations were considered. However, although there was a fairly large range, in a future investigation, a far greater number of substrate concentrations between those already recorded should be tested reducing the extent of any anomalies on a graph where the line of best fit must be drawn.
In this investigation each reading was repeated so that an average rate of reaction for each substrate concentration could be calculated. This could be improved by repeating the reading more frequently thus reducing the extent of any anomalies further, once averaged.
Finally I think had smaller quantities of hydrogen peroxide solution and celery solution been used a greater range of values could have been tested giving a more extensive graph.
Overall I think that the experiment was completed to the best of its potential, and that the results were sound, and accurate enough to prove my theory, that as the substrate concentration increases so does the volume of O2 produced because the rate of reaction has increased. This is because there are more molecules to reactive with the active enzyme sites, which means there are more collisions resulting in more successful reactions, which produces more O2.. The results were all fairly accurate, the only ones which could be slightly inaccurate, are those which were taken at concentration 5cm3 hydrogen peroxide/ 5cm3 distilled water. This is because as the concentration was quite high and the reaction took place very quickly, some O2 might have escaped into the burette after 1 minute had passed. The results for this concentration are very similar though which suggests there were not too many mistakes.
Extending the investigation:
If I were to extend this investigation I would repeat the same experiment but keep the substrate concentration the same and instead change the enzyme concentration. The enzyme concentration experiment should have similar results to this experiment based on the collision theory and providing there were a sufficient number of substrate molecules for the enzyme to react with. Also if the equipment and variables were all kept the same except obviously the enzyme concentration, then the results could be compared to this experiment, which would show whether changing the enzyme, or substrate concentration had a greater effect on the rate of reaction.
I would also investigate the catalase content of other foods, such as liver and potato, which contain the enzyme catalase. I would use the same method as I did for the celery, as this would make the test fair. I would blend the same quantity, 100cm3, of potato or liver with 100cm3 of water for 15 seconds. This will break open the cells and release the enzyme. I would then put 10cm3 of the solution into a chronicle flask and add 10cm3 of hydrogen peroxide solution. This would be timed for 1 minute and the volume of oxygen produced recorded using a burette. The experiment would be repeated using the 5-substrate concentrations used for the celery solution. The experiment would be repeated 3 times for each concentration so that the average volume of oxygen produced could be recorded. The rate of reaction would also be worked out using the equations explained for this investigation. This data would be presented on a graph, which could then be easily compared to the reaction rate of the celery solution. From the conclusions drawn from the graph it should be possible to tell whether liver, potato or celery contained more catalase.
I would also like to investigate the effect of temperature on the break down of hydrogen peroxide using celery tissue to supply the enzyme, catalase. This would show greater depth into the topic and hopefully provide more conclusive results as to the conditions needed for the optimum yield of the products given off when hydrogen peroxide reacts with catalase.
From background research it is possible to discover that a rise in temperature increases the rate of enzyme-controlled reactions; a fall in temperature slows them down. In many cases a rise of 10oC will double the rate of reaction, but above 50oC the enzymes, being proteins, are denatured and stop working.
Knowing this it is possible to compose an experiment which will test if the above information is true for the enzyme, catalase and work out the temperature at which the rate of reaction is highest.
Prediction
It is predict that the enzyme catalase will only work at temperatures above 10oC and below 50oC. The optimum reaction rate will lie somewhere between 30oC and 40oC. As catalase is an enzyme, which is used in the body, it is likely to have a high yield at 37oC, which is body temperature.
The predicted graph shows how the rate of reaction doubles as the temperature increases by 10oC and how below 10oC and above50oC the catalase is denatured and does not function.
Method
The apparatus would be set up in the same way as the experiment for substrate concentration only using an ice bath or Bunsen burner to get the celery solution to the required temperature.
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Blend 100cm3 of celery with 100cm3 of water in a food processor.
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Use 10cm3 of the celery solution and place in a chronicle flask.
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Cool to 10oC using an ice bath.
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Put 10cm3 of hydrogen solution into the chronicle flask.
- Leave to react for 1 minute.
- Record the results.
Repeat for 20, 30, 40, 50 and 60oC. Work out average O2 produced for each temperature and rate of reaction. Draw graphs using the results collected. From the graphs conclusions can be drawn as to the nature of catalase and the best conditions needed for the fastest reaction rate.