Influence of Temperature on the Activity of Potato Catalase

Influence of Temperature on the Activity of Potato Catalase
Hypothesis That the higher the temperature the higher the reaction rate of potato catalyse to a point were denaturing occurs in the enzyme and the reaction rate of the potato catalase drops off. Prediction The rate of Catalase activity will be faster at higher temperatures until a point, because at higher temperatures there are more chances of collisions between the enzyme?s (Catalase) active site and the substrate (hydrogen peroxide). However the rate depends on the active site being able to join with the substrate, and at higher temperatures the enzyme can be denatured, which changes the shape of the active site which thus prevents the reaction from happening. At first, as the temperature increases the activity of the Potato catalase also increases this is because the collision rate of the enzyme with the hydrogen peroxide is increased. The reason for this is that as you increase the temperature of the enzyme and the substrate molecules you also increase the energy in the form of kinetic energy which they possess.
Because the enzymes and substrate molecules are moving faster the chance of them colliding in a certain time and area is increased. Thus the chance of the hydrogen peroxide molecules binding with the Catalase molecules active site and reacting is higher. However after a certain point increasing the temperature will begin to hinder the activity of the potato catalase. The reason for this is that the energy being supplied to the enzyme begins to effect its stability. The energy supplied begins to make the atoms which make up the enzyme move and vibrate rapidly, at a certain point the enzyme atoms are vibrating at such a rate that the bonds that hold the active site together, such as the disulphide and hydrogen bonds are broken and the shape of the active site changed. This is vital as the Hydrogen peroxide substrate can no longer bind with the catalase enzyme and react. The activity of the catalase is dependent on the balance of increasing the temperature and increasing the reaction rate and denaturing the enzyme. Thus the temperature that produces optimum activity of the Catalase is the temperature just before the enzyme begins to denature. This is because the collision rate is at its maximum without the denaturing effect coming into play. It is possible to test the activity of the potato catalase with hydrogen peroxide because potato catalase breaks down hydrogen peroxide into oxygen and water. The oxygen gas can therefore be collected to ascertain the activity of the Catalase at certain temperatures. I have produced a predicted graph of how the activity of potato catalase will change through increasing temperature. There are a variety of factors which effect the break down of hydrogen peroxide by the enzyme catalase which I must consider to ensure that my experiment produces fair and reliable results: Ÿ The concentration of substrate (hydrogen peroxide), the higher the substrate concentration the higher the reaction rate. I will use hydrogen peroxide ?volume 20? because it will produce a reaction that will not be restricted by substrate concentration and will operate at a level sufficient to measure easily, also using a volume higher then 20 could be dangerous . Ÿ Enzyme concentration and volume, The higher the enzyme concentration and volume the faster the reaction due to higher collision rate and higher number of active sites available for reaction. As the enzyme catalase that I am using is held within the potato the concentration and volume of catalase depends on the potato used. Because of this I will be trying to use the same potato on each run through of all the temperatures in the experiment, also when I do have to use a new potato (I.e. if the experiment is done on more than one day) then I will use the same type of potato (I.e. the same breed etc). Ÿ Surface area of potato, the higher the surface area the higher the activity of potato catalase the reason for this is that more enzyme comes in contact with the hydrogen peroxide. To keep this constant I will use ten discs of potato 1cm diameter and 1mm thick in each experiment to keep surface area and mass of potato constant this will hopefully keep enzyme volume and concentration constant as well. Ÿ The temperature of the reaction also has a effect as I have discussed already. I will be investigating this effect be doing the reaction at a range of temperatures (10C, 20C, 30C, 40C and 50C). Ÿ Time enzymes are exposed to temperatures, enzyme denaturing is time dependant so enzymes that are exposed to high temperatures for a short period of time will be less denatured than those exposed for a long time. To combat this I will equilibrate the reactants in the experiment at the desired temperature in a water bath for 5mins to ensure that the enzyme and reactants are at the right temperature during the reaction. Ÿ pH, each enzyme works differently at different pH values. I found in a pre-test that catalase works best at pH7. Because of this I will be using a buffer to maintain the pH at 7. I have decided to use this pH to one produce a good range of results and also ensure that pH does not restrict the reaction. Procedure 1. Using a cork borer cylinders of 10mm diameter are taken from a potato and cut into discs of 1mm thickness and then placed into a Petri dish of distilled water (so that the potato does not dry out). Enough discs for one run of experiments are cut (I.e. 10 for each temperature thus 50 discs are produced) 2. Apparatus shown below is set up : 1. The position of the manometer fluid meniscus is marked on the manometer tube as is a point 5cm above that point. 2. Removing the bung 5cm3 of citric-acid phosphate buffer of pH7 is added using a 5cm3 syringe. 3. Add ten of the 5mm diameter 1mm thick potato discs to the boiling tube. 4. 5cm3 of hydrogen peroxide ?volume 20? is added to a separate test tube. 5. Both the boiling tube and the test tube are then added to a water bath of desired temperature and allowed to equilibrate before they are reacted together. 6. The 5cm3 of hydrogen peroxide is then added to the boiling tube containing the buffer and potato discs and the bung replaced ensuring a seal is created. The reason for adding the hydrogen peroxide last is that it ensures that the reaction does not begin before the bung has been replaced. 7. The boiling tube should be gentle agitate to ensure that the catalase and hydrogen peroxide are mixed together and begin to interact. 8. Once the bung has been replaced the clip is closed and the stopwatch is started and the time taken for the manometer fluid to rise 5cm is recorded. 9. Once the manometer fluid has risen 5cm the time taken is recorded and the clip released so the fluid is returned to its original position. The test is then repeated three times at each temperature an average time taken recorded. The reason for this is so that anomalous results can be spotted . If the test was not repeated then it would be impossible to spot an anomalous results and thus any conclusions would be based on flawed information. 10. Express results as rates of reaction , in arbitrary units , by dividing 1000 by time taken in seconds for 150mm3 of gas to be displaced ( I.e. the time taken for the manometer fluid to rise 5cm in a manometer tube of 3mm diameter). Safety The hydrogen peroxide that I am using is a dangerous chemical to work with due to its corrosive properties because of this there are several safety implications that must be adhered to during the experiment. Ÿ During usage eye protection should be worn at all times. Ÿ If the hydrogen peroxide is digested then the mouth should be washed out with water and medical attention sort. Ÿ If the liquid gets into the eye then flood the eye with a gently running tap for 10 minutes and then seek medical attention. Ÿ If the hydrogen peroxide is spilt on clothes or skin then the affected area should be flooded with water, remove contaminated clothes and soak. If blistering on the skin occurs or large area affected then seek medical attention. Ÿ If the hydrogen peroxide is spilt in the laboratory it should be covered with mineral absorbent and cleared into a bucket (must be wearing gloves and eye protection). Dilute with water and wash liquid down the foul water drain. Ÿ During disposal the hydrogen peroxide should be diluted with water in a bucket and washed down the foul water drain. Ÿ Hydrogen peroxide should be stored in a dark brown bottle and care must be taken when removing the cap as it is possible that pressure may have built up. It should also be stored with other corrosive liquids and should only be diluted before use due to the fact that the inhibitor is also found as a dilute. Temperature (0C) Time taken (seconds) 1st trial Time taken (seconds) 2nd trial Time taken (seconds) 3rd trial Time taken (seconds) 4th trial 10 42.85 43.73 59.23 59.19 20 31.90 31.86 50.10 49.97 30 20.17 19.98 39.91 39.86 35 13.08 12.97 32.86 32.71 40 16.08 16.05 35.34 35.30 45 29.95 29.89 48.93 48.75 50 57.02 56.93 78.02 77.94 60 stopped stopped stopped stopped Summary Table I have created an average of the time taken for the 150mm3 to be displaced by adding up the time taken for the four trials and dividing by 4. e.g. 42.85 + 43.73+59.23+59.19 = 51.25 4 I then created an average reaction rate by dividing the gas produced (150mm3) by the time taken for that gas to be produced. e.g. 150 = 2.93 (2.d.p.) 51.25 I decided not to use error bars on my graph because I measured time taken with a stopwatch that was accurate to 100th of a second so I decided that the possible error using this method was not significant. Temperature (0C) Average time taken (seconds) Average reaction rate mm3/second (2.d.) 10 51.25 2.93 20 40.96 3.66 30 29.98 5.00 35 22.91 6.55 40 25.69 5.84 45 39.38 3.81 50 67.48 2.22 60 stopped 0 Analysis As temperature was increased the reaction rate of the potato catalyse also increased to a optimum temperature when reaction rate was at its highest until denaturing in the enzyme occurs and the reaction rate of the catalyse drops off. Temperature (0C) Average reaction rate mm3/second (2.d.) 10 2.93 20 3.66 30 5.00 35 6.55 40 5.84 45 3.81 50 2.22 60 0 The results from trails 1 and 2 and 3 and 4 are significantly different with every result being an average of around 20 seconds different. This suggests that there is something anomalous about the variation in these results. The reaction rate of the Catalyse increases in a fairly straight line between 10 degrees and 35 degrees were an optimum temperature is reached. The optimum temperature from my results lies between 35 degrees and 40 degrees. However from my results I would expect that the optimum lies closer to 35 degrees because that is the temperature were reaction rate is highest. The reaction rate of the Catalyse then begins (after 35 degrees) to drop off also at a fairly constant rate until the reaction rate is reduced to zero at 60 degrees. This suggests that the catalyse enzyme begins to denature after the optimum temperature is reached around (35-40 degrees). At first the higher the temperature the more oxygen that is produced because there is a higher chance of the substrate hydrogen peroxide binding with the potato catalyse thus producing oxygen. The reason for this is that at higher temperatures both the hydrogen peroxide and the potato catalyse are supplied with more energy. This means that they have more kinetic energy and are moving around at higher speeds. This means that there is a higher chance of collisions between the substrate and the enzyme and thus more chance of enzyme-substrate complexes being formed which in this case produces oxygen. However there is a point were the increasing temperature causes a problem for the action of the potato catalyse. This is because there is so much energy being provided by the heat that the individual atoms which make up the enzyme catalyse are vibrating rapidly. This can in turn cause the bonds between the atoms in the enzyme such as the hydrogen and disulphide bonds are broken. This means that the shape active site of the enzyme is changed. And because the enzyme active site is specific to the substrate the enzyme can no longer bind with the substrate because of the change shape and so they cannot react. The optimum temperature were the highest reaction rate is found were the balance of higher temperatures with high collision rates and denaturing occurring. The optimum temperature is found were the highest collision rates can be achieved but were denaturing does not come into play. Evaluation The results that I have obtained I think are fairly reliable despite some problems. I also think it is perfectly reasonable to say that clear conclusions can be made about the influence temperature on potato catalyse from my results. I decided not to use error bars on my graph because I measured time taken with a stopwatch that was accurate to 100th of a second so I decided that the possible error using this method was not significant. In my results there is a fair amount of variation in respect to the first two trials and the second two trails despite this I think that my results are still fairly reliable. The reason for these anomalous results is due to the fact that I was forced to change the potato I used after two trials because I did not have time to do three trials on one potato. My original plan was to test until I found three concordant sets of data and at first I believed the 3rd trial to be an anomalous result. Because of this I repeated the test and done a further trial at each temperature however using the same potato as the 3rd trial. What I found was that by using the same potato I got concordant results. From this I deduced that different potato?s must vary something about the potato catalyse. I concluded that the most sensible answer was that different potato?s had varying concentrations/volumes of catalyse. However what I did notice was that despite the fact the reaction was slower in the second two trials the rate of increase/ decrease when the temperature was varied was concordant between all three trials. For this reason I felt that it was appropriate to use all the trials in my average because the change in the reaction rate was the more important feature of the experiment than the actual reaction level at different temperatures themselves. Also by using all four trials I can make more general comments about potato catalyse in general rather than just the potato catalyse found in one potato. The problem with having to change the potato highlights the problem which I had in controlling the concentration and volume of potato catalyse. The reason for this problem was that I had no way of knowing, using my method, what concentration of or volume of potato catalyse I was using. I was having to rely on the fact that by using the same volume of potato each time that I was also keeping the concentration and volume of potato catalyse the same. However there could have been variation within each potato and within different potato?s which would have also cause variations in my results. Another problem was keeping the temperatures used constant during tests and between trials. The problem was that I wanted to test so many different temperatures that I could not feasible use a electronically controlled water bath to do each test. The result was that my using a Bunsen burner method it was hard to get exact temperatures and maintain them throughout the experiment. This may have meant that the test were not exactly done at the right temperature this may have cause problems with the results collected and cause variation between results. To improve the experiment it may be better to, rather than use potato?s as a source of the catalyse in the experiment to use a solution of catalyse of some kind which the concentration and volume of could be controlled. This would make it much easier to control the volume and concentration of the potato catalyse which would result in a much fairer test. Also I would change the method of the test to include the temperatures to be controlled by means of an electrical water bath which keeps the temperature of the water bath constant using a thermostat. This would mean there would be less variation with the temperature of the thermostat and thus less variation and more reliability in the results. Overall I feel that the results that I have gained do support the hypothesis that I laid out as they clearly show that the reaction rate of the potato catalyse increasing as temperature to an optimum point were after the reaction rate dropped off due to denaturing.

Influence of Temperature on the Activity of Potato Catalase 8.1 of 10 on the basis of 2952 Review.