In my experiment I plan to seal the tube around the bung and the gas cylinder as I noticed some gas escaping. I think that Vaseline will be suitable for this.
I found the liver extremely difficult to cut but, unfortunately, I cannot see a way around this problem, as very sharp knives are not available for my use.
Another problem I encountered is that I had to be very quick when adding the liver, copper sulphate and putting the bung in place as the reactions began immediately and a considerable amount of gas escaped in my delay during the first few tests.
Variables:
The variable in this experiment is copper sulphate solution because I wish to see how an increase in this concentration will affect the enzyme reaction between catalase and hydrogen peroxide.
I am using distilled water in this experiment to make the total volume of liquid the same in each case. This, therefore, must be measured carefully in an appropriate measuring cylinder. An incorrect amount of distilled water would result in inaccurate results.
It is important that other factors remain constant:
- Catalase - The amount of catalase must be kept the same if the experiment is to be a fair test. If there is an increase in catalase, there will be an increase in the initial reaction rate; if there is a decrease in catalase, there will be a decrease in the initial reaction rate. The more enzyme present, the more active sites will be available for the substrate to slot into. I will ensure that the amount of catalase is kept the same by carefully cutting the liver so that the surface areas are all the same. However, the most reliable method available to me is using a scalpel but this does not guarantee 100% efficiency.
- Hydrogen peroxide - It is important that this remains constant as an increase in substrate concentration will result in an increase in the initial reaction rate as the more substrate molecules there are around, the more often an enzyme’s active site can bind with one. I will ensure that this remains constant by using as small a measuring cylinder available so that the reading is as accurate as possible.
- Temperature - It is important that temperature is kept constant as an increase in temperature is likely to alter the reaction rate. The solutions will all have been stored at room temperature so the temperature should be consistent throughout the experiment.
- pH level - this is a major factor affecting the rate of an enzyme controlled reaction. All enzymes have an optimum pH (this is usually around 7 with most enzymes) but this is not a factor concerning this reaction as we will not be altering the pH level throughout the experiment. I will have to be careful that only the proposed solutions get into the conical flask as any alien substances may vary the pH.
Justification:
I believe that my hypothesis will be proved correct due to a number of reasons. Firstly, my research shows that copper sulphate is an inhibitor of the enzyme catalase, so it would be expected to reduce the rate of reaction. Secondly, my preliminary experiment supports this theory and although it is not 100% accurate, I believe that the rough curves shown on the graph clearly outline the results I expected.
I believe that I will achieve satisfactory results with the apparatus I have chosen but there are considerable limitations in the equipment available. It is imperative that my strategy is precise and reliable in order to obtain accurate results. It is unfortunate that a wider range of apparatus is not available for use but I believe that I have selected the most appropriate out of the selection.
A more accurate way of obtaining pieces of liver with exactly the same surface area could be achieved with the use of a sharper knife, or a very sharp borer but unfortunately I will have to make do with what I have. Similar surface areas are attainable but to be more precise I would have to have more specialist facilities to hand.
The gas cylinders available are quite worn and are quite thick. If they were thinner, the readings would be more accurate. Also, there is a risk of some of the gas escaping through gaps where the rubber tubing attaches to the gas cylinder and to the bung. This can be overcome with the use of a Vaseline seal.
I plan to measure the amount of gas produced at 20-second intervals. I feel that this is a suitable time as, for example, 30 seconds may prove to be too long and 10 seconds may be too short (the reactions for the lesser concentrations of copper sulphate solution will be much faster than those for the greater concentrations: 10 seconds would be more suitable for the former and 30 seconds for the latter, so 20 seconds would be the mid-range).
Despite my best efforts to ensure that a minimum amount of gas is allowed to escape between the addition of the liver to the H2O2 and the placement of the bung, it is inevitable that some will escape. This will adversely affect my results slightly but I think I can still obtain satisfactory results, which will prove my hypothesis.
Despite certain unavoidable blemishes to the method, I believe that an adequate curve will be achievable when it comes to plotting the results.
Risk Assessment:
- Extreme care must be exercised whilst cutting the liver as it is very easy to cut your fingers – especially as the liver is so difficult to cut.
- As with all chemicals, the hydrogen peroxide and copper sulphate must be handled with caution. Goggles should be worn to protect the eyes and hands should be washed after the experiment to prevent the entry of these substances into the body.
- Glassware must be handled with care.
Sources:
I obtained the information used in the introduction from several biochemistry sites on the internet (including various homepages and mrmicrobe.com) and from my AS level textbooks. I believe this information to be reliable as it was consistent over several sources.
I also obtained details of a similar experiment from an American student’s homepage on the internet and I used this information to help formulate my method. I cannot, however, take this information as being completely accurate as it was compiled by a student of a similar age to myself and there was no mark attached to her investigation. There was, however, some useful insights into the inhibitory effects of copper sulphate on the reaction between hydrogen peroxide and catalase. Whereas the source of catalase in my experiment is liver, she used potatoes. Therefore, I think that our rate of reaction/time curves will look similar but the figures will be very different.
Her experiment was as follows:
Apparatus: 50ml 1% Copper Sulphate solution; 50ml 20 volume hydrogen peroxide solution; scalpel; ceramic tile; forceps; distilled water; Petri dish; manometer; beaker; 2 x 5cm³ syringes; stopwatch; potato
Method: Using a cork borer, cut out a slice of potato at least 5cm long. Slice the cylinder into discs 1mm thick using a scalpel and place them in a Petri dish of distilled water. Approximately 60 discs will be required. Assemble the manometer, taking care not to damage the glass tubing when inserting the rubber tube on it. Remove the bung from the boiling tube and place 10 potato discs into it. Add 5cm³ hydrogen peroxide followed by 5cm³ distilled water into the boiling tube using a syringe. Replace the bung immediately, making sure that the airtight seal is achieved. Note the time. Mark on the right hand manometer tube the position of the meniscus and draw a thin line exactly 5cm above it. Gently shake the boiling tube to fill all of the available active sites and time how long it takes for the manometer fluid to rise the 5cm up the right side of the manometer. Open the control clip at the top of the rubber tubing so that the manometer fluid returns to its original position. Close the clip again and repeat the first reading. Do this for a third time and calculate the average time taken to travel the 5cm distance. Remove the bung and thoroughly rinse out the boiling tube. Repeat the experiment for the volumes of hydrogen peroxide, copper sulphate and water outlined in the table below.
Results:
Evaluating Reliability of Source:
This curve is what I would have expected from the addition of copper sulphate to the reaction. In this respect this evidence does assist me in my investigation but as our methods differ considerably and I am not certain about the reliability of her method, that is the extent at which I can use her information. It is useful in the sense that it reinforces my hypothesis.
Analysing Evidence & Drawing Conclusions
The method provided differs somewhat from my plan:
Therefore, experiment A represents a CuSO4 solution of 0M, experiment B represents a CuSO4 solution of 0.25M, experiment C represents a CuSO4 solution of 0.5M, experiment D represents a CuSO4 solution of 1M, experiment E represents a CuSO4 solution of 1.5M and experiment F represents a CuSO4 solution of 2M.
Referring to each experiment by its allocated letter will simplify the format of my results (i.e. A2 indicates the second repeat for a concentration of 0M, F3 indicates the third repeat for a concentration of 2M, etc.).
- 6 pieces of liver with masses of 1.5g are weighed out making sure that the surface areas are as similar as possible.
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Each piece of liver is put into each CuSO4 solution and left for 10 minutes.
- As apparatus are set up as shown below:
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A piece of liver is put in the conical flask, then 2cm³ of H2O2 is added and the bung replaced.
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The volume of O2 released is measured every 30 seconds for 2 minutes.
The Results from My Experiment:
Average Values Obtained from my Results:
I also obtained results from fellow students:
#1
#2
I have worked out an overall average using all of the above results:
Note: - All measurements are written to one significant figure.
Comparing Results:
The results obtained from this experiment are all quite different yet they show similar trends: the amount of gas produced decreases as the concentration of the CuSO4 increases; the graphs all show similar curves.
I now plan to work out the gradients from all the lines on my graphs. The formula for this is vertical distance (y) divided by the horizontal distance (x). I have plotted a line of best fit.
The above graph shows how, for each concentration, the gradient decreases with time and how it decreases as concentration increases. This means that the curves became less and less steep for each increasing concentration. Therefore, the higher the concentration of CuSO4, the more the rate of reaction decreased.
An enzyme-controlled reaction will become increasingly slower until it stops completely. This is because when the enzyme and substrate are first mixed, there is a large number of substrate molecules. Before long, practically every enzyme molecule has a substrate molecule in its active site and the reaction rate has reached its maximum. As more substrate is converted into product there are fewer substrate molecules to bind with enzymes. The reaction will begin to slow down until no substrate remains and then it will stop completely.
During my initial research I discovered that CuSO4 has an inhibitory effect in the reaction between H2O2 and catalase and my results support this. Inhibitors block or alter the shape of the active site on an enzyme thus preventing a substrate molecule from binding to it. The greater the amount of inhibitor, the less likely it is that substrate molecules will collide with a ‘free’ active site. This corresponds with the decrease in reaction rate as more CuSO4 was added.
Although all my graphs are different the essential similarities are obvious. Experiment A produced the most gas, followed by experiment B, then C, and so on. The initial rate of reaction was faster for the lower concentrations of CuSO4 and then levelled off sooner; the higher concentrations CuSO4 began slower and continued gradually rising. This is due to the fact that CuSO4 is an inhibitor for the enzyme catalase. At a lower concentration of CuSO4 there are more free active sites for substrate molecules to bind with than at higher concentrations. The reaction for lower concentrations began quickly as many substrate molecules could bind with active sites but as the reaction progressed, the substrate began to diminish and so the reaction became slower and eventually stopped. At higher concentrations of CuSO4 there are fewer active sites available for substrate molecules to bind with and therefore the reaction progresses slowly. If left for a longer period of time, the reaction would eventually stop but it would take much longer for all the substrate molecules to be converted as there would be fewer working active sites. There may even be a situation where all the enzymes are bound to an inhibitory substance and so no reaction between enzyme and substrate can occur at all.
Unfortunately, I was unable to time each test for longer than 2 minutes in the time allowed.
I believe that the overall average results are the most reliable as they cover the widest range of results. It is these that I will use when analysing my findings.
My second range of graphs show how the total volume of gas collected is relative to the concentration of the CuSO4 solution. I have drawn a line of best fit for this graph as I believe it should be straight (i.e. gas produced is directly proportional to the concentration of CuSO4 solution). My reason for this assumption is simple: common sense tells me that a 1M CuSO4 solution will produce half as much gas as the control experiment (0M) and a 2M solution of CuSO4 will produce twice the difference between 0M and 1M. This is drawing from my knowledge on enzyme inhibition as described above.
The method was not wholly reliable, as I will elaborate on in my evaluation, but the results obtained do show a trend indicating a straight line.
Conclusion:
The results of my experiment show that as the concentration of CuSO4 increases, the rate of the enzyme-controlled reaction decreases. The maximum rate of reaction measured was in the control experiment (A). In this reaction there was no CuSO4, only catalase and hydrogen peroxide. In each case from then on, as the amount of CuSO4 increased, the rate dropped significantly. The lowest rate of reaction was calculated in experiment F.
My hypothesis: Based on my research, I believe that copper sulphate is an inhibitor of the reaction between the enzyme catalase and hydrogen peroxide.
I predict that my results will show that the greater the concentration of copper sulphate, the greater will be the inhibition and thus the slower the rate of the enzyme controlled reaction
My results therefore suggest that copper sulphate is indeed an inhibitor of the enzyme catalase since its increasing concentration causes a progressively lower rate of reaction. I can also deduce that it could be a non-competitive inhibitor, as suggested in my hypothesis, attaching itself to the enzyme away from the active site and thus preventing the catalytic action of the enzyme. The more inhibitor molecules there are, the more enzyme molecules altered in this way.
Types of Enzyme Inhibition:
Taken from Cambridge Advanced Sciences ‘Biology 1’
Competitive inhibitors: ▪ have a shape similar to that of the enzymes normal substrate
▪ fit temporarily into the active site
▪ substrate cannot enter if the inhibitor is in the active site
▪ the inhibitor and substrate compete for the active site
Non-competitive inhibitors: ▪ bind to a location on the enzyme other than the active site
▪ have a different shape to the normal substrate
▪ cause a change in the shape of the active site thus preventing
substrate from binding
Reversible inhibitors: ▪ not permanently bound to the enzyme (when they leave, the
substrate is free to enter the active site)
Irreversible inhibitors: ▪ bind permanently to the enzyme or permanently alter the
active site (the substrate will never be able to enter the active
site
Evaluating Evidence and Procedures
Although the results obtained are not entirely concordant, they are satisfactory enough to enable me to test my hypothesis and draw an accurate conclusion.
There were no obvious anomalous results on the Volume/Time graphs but the Volume/Concentration graphs all appeared quite scattered. I drew a line of best fit, which proved my theory that the volume of gas collected would be directly proportional to the concentration of CuSO4 but there were obviously a few experimental errors made to cause these anomalies. I have only circled the most severe anomalies, which I feel would make a considerable difference to my results as few readings sit exactly on the line. In reality, every result that is not entirely concordant with all the others should be considered anomalous but although clear trends can be observed, none of the results seem to be 100% concordant.
Comparing Different Sets of Results:
For the purpose of making this experiment a fair test I have collaborated my results with those of two other students. All the results obtained show a general trend: as the concentration of CuSO4 increases, the amount of gas produced decreases. All the graphs acquired from these results show similar curves (with the control experiment having the fastest reaction and levelling off first and the reaction with the 2M CuSO4 solution having a very slow rate of reaction – all graphs clearly show that as concentration increases, rate of reaction decreases. All results obtained support my hypothesis.
My measurements appear considerably higher than those of the 2 comparative experiments. I suggest that this is most likely to be because the size of the liver I cut for my experiment was larger (and therefore had a larger surface area) than that of my fellow students.
I calculated an overall average result compiled from all three experiments. I feel that this practice has enabled me to achieve a higher level of accuracy and the graph drawn from these results is what I expected to see prior to the experiment (i.e. it is more concordant with my hypothesis than the individual results). To gain an even higher degree of precision, I would have included more sets of results in my analysis and then the average values would have been more accurate.
As previously described in the analysing section, I believe that there is a relationship between the concentration of CuSO4 and the volume of gas produced (they are directly proportional to each other). This is what I think I would witness from a graph of 100% accuracy:
Evaluating Method:
- I believe that the greatest inaccuracy was caused by an inadequate method for measuring the surface area of the liver. We were able to weigh it accurately on the balance but in this experiment it is the surface area that counts. The surface area must remain constant, as a larger surface area would result in an increase in rate of reaction because there would be a greater amount of collisions between enzyme molecules and substrate molecules. I think that the method I devised in my plan would have worked better than the method provided. A further improvement would have been to use an instrument such as a very sharp cork borer.
- The apparatus we used were quite primitive and I feel that this affected our results. For example, when the rubber tube is submerged to put the end in the measuring cylinder, it fills up with water: this water must be pushed out by the gas being produced before any of it can be measured. It is likely that the amount of water in the tube was different every time, so this was not even a constant. A more precise piece of apparatus would have been a gas cylinder (with Vaseline around the seals of the rubber tube to prevent leakage) or a manometer, which measures pressure. These changes would have resulted in a greater level of accuracy in my results as the issues raised above (with reference to the method used) would have been irrelevant.
- Clean glassware was not always available throughout my experiment so I had to make do with thoroughly washing out the same conical flask after each use. Any liver residue left inside would have increased the overall surface area and thus preventing my experiment from being a fair test.
Evaluating Procedures:
- As there was limited time in which to complete the experiment, some aspects of it may have been rushed. I may have read the measuring cylinder wrong in my haste or I may have not started the timer early enough or started it too early. However, I think that the main source of error was in the measuring of surface area of the liver. Had there been more time in which to complete the experiment, then I feel that more time could have been taken ensuring that all pieces of liver were of the same surface area.
- 1.5g of liver was quite a large block and the 2cm³ of hydrogen peroxide did not completely cover it when in the conical flask. Therefore, only a fraction of the surface area of the liver was reacting with the hydrogen peroxide and this would have varied from test to test. A way of improving this would be to replace the conical flask with a test tube or boiling tube as the liver would be submerged in the hydrogen peroxide. Alternatively, a smaller amount of liver could have been used, or several small pieces.
- Each person’s results that I used in the analysis differed quite a lot. This is because, in the method we were told to “weigh out 6 pieces of liver with a mass of 1.5g, making sure that the surface area of the pieces is as similar as possible”. As there was no standard set for surface area, each person was cutting different shaped pieces of liver. It could not be expected for all our results to be entirely concordant with each other’s.
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As I know from my initial research, temperature has a considerable influence over the rate of an enzyme-controlled reaction. All enzymes have an optimum temperature at which it catalyses a reaction at the maximum rate. At lower temperatures, the molecules have little kinetic energy and therefore there are fewer collisions. As temperature plays such an important role in enzyme-controlled reactions it is imperative that it remains constant throughout an experiment. Although the experiment was done under stable conditions, the temperature was not carefully monitored. Variations in temperature would not have been detected and in this respect, the experiment was not a fair test. There were several occasions where a change in temperature could have occurred: at one point I ran out of liver and the laboratory technician defrosted some more for me. This liver could not have been at exactly the same temperature as the liver I was using before. I should have ensured that there was a plentiful supply of liver that had been left to acclimatise for a sufficient amount of time before I used it. Also, as I had to keep using the same glassware, a significant temperature change could have occurred through rinsing in cold water. Had there been an adequate supply of glassware, this problem would have been eradicated.
Was I to repeat this experiment I would make significant changes as described above in order to obtain a higher degree of precision in my results. However, despite there being considerable room for improvement in my method, I am confident that the results I obtained were sufficient in order to prove my hypothesis. The graphs formulated from these results showed trends that I expected to see and the trends observed over the whole range of graphs were consistent. They clearly show the effect that copper sulphate has on the action of the enzyme catalase despite errors being made.
In order to find out exactly what type of inhibitory effect copper sulphate has on the action of the enzyme catalase, further experimental procedures would have to be followed. If the rate of reaction increased when more substrate was added, it would be a competitive or non-competitive reversible inhibitor; if the rate of reaction did not increase when more substrate was added and merely continued to slow down, it would be a non-competitive irreversible inhibitor. Further experiments beyond my scientific knowledge would be required to differentiate between a non-competitive and competitive reversible inhibitor
Had all the variables been precise throughout the experiment I think that everyone’s graphs would have shown identical curves with the same values for each reading throughout a range of repeats. The errors made were due to several factors: insufficient planning prior to performing the experiment, personal error, inappropriate equipment, insufficient repeats etc.
