Investigating the effects of temperature on an Enzyme Catalase.

• Instead of measuring the length of the potato I will weigh it to two decimal places.
• I will make sure that all five bits of potato have the same surface area, because otherwise there will more catalase exposed, therefore creating a larger rate of reaction.
• I am going to make sure there is no trapped air inside the syringe, before I do the experiment.
• I will make sure all the potato skin has been removed before proceeding with the experiment, as it will slow down the rate of reaction.

Prediction to show how changing my independent viable will effect the rate of reaction.

Temperature

This graph shows the temperature rate of reaction.
As the temperature increases both the enzyme and substrate gain heat, causing them both to move around more, this means that the substrate makes more successful collisions with the enzymes active site, thus the rate of reaction increases, once the optimum temperature is reached the rate of reaction falls rapidly, as shown on the graph above.

‘The collision theory’

Too much heat means collisions between the enzyme and substrate are too strong. These strong collisions mean that the enzymes can denature, causing the active site to change shape, meaning the substrate will not fit in the active site. If there is no active site, then reactions will not be able to take place.

Another theory is the lock and key theory, if we consider the enzyme as the lock and the substrate the key (Figure 1) - the key is inserted in the lock, is turned, and the door is opened and the reaction occurs.

Figure 1

 

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To increase the accuracy of my investigation I will:

• Use the smallest syringe available (10 ml), when collecting the oxygen to get a more precise result.
• Take my time when using the pH buffer and hydrogen peroxide to get the most accurate amount in the boiling tube.

Thanks to a preliminary investigation that I carried out, I can use the results, as shown below, to predict what is going to happen in my full investigation:

Results:

    Temperature                                                     Rate of reaction cm3/ min

3        0.3

17        1

25        1.2

33        1.2

50        0.8

I recorded my results, in the preliminary investigation, as cm3 per 3 minutes so to convert to, rate of reaction per minute I used the formula below:

 

Oxygen collected ÷ Time taken

   For Example                                  3cm3 ÷ 3 minutes

The preliminary investigation helped me set up my full investigation because, I can improve on the mistakes I made before and make sure the same thing does not happen again. I now know that using the smaller 10 ml syringe will make my results more accurate than if I was to use the larger one. I know that weighing my potato is much more accurate than if I was to measure the length of the potato.

Two factors I will find hard to control will be the temperature and the enzyme concentration.

• The cold temperatures will be hard to control because the water will start heating up to room temperature. I will control the temperature by using a thermometer and adding appropriate amounts of ice to keep the temperature the same.
• The enzyme concentration will be hard to control because the weight of the potato will be very hard to keep the same for all five pieces, however I will make sure the weight of each piece of potato does not go below 2.70 grams or above 2.72 grams.

Plan of action for my full investigation.

First I will bore out five cylinder pieces of potato on a square white tile using a metal borer. I will then take off the thin layer of potato skin using a sharp metal razor, once the skin is off I will then roughly cut all five pieces of potato so they are about 5cm long. Once they are all roughly the same size I will then weigh them making sure they all weigh no less than 2.70 grams and no more than 2.72 grams.

Next I will fill a large beaker with normal water from the tap, then I will set up the apparatus (a detailed plan below shows how to set up the apparatus). When the apparatus is set up and ready to go I will prepare five foam cups and fill them with the following:

Cup 1- Water measuring   0oc

Cup 2- Water measuring   15oc

Cup 3- Water measuring   35oc

Cup 4- Water measuring   60oc

Cup 5- Water measuring   80oc

A kettle will heat the water where appropriate. The cups will then each be placed in a plastic holder. Once the cups have been heated to the appropriate temperature, I will then place 5cm3 of Hydrogen peroxide, which must be kept constant, and 5cm3 of ph7 buffer, the most neutral pH, into each of the boiling tubes. When I have checked the set up and am satisfied with it, only then shall I carry on with the investigation. I will drop a piece of potato into the boiling tube, put in the rubber bung and start timing the reaction. Once the three minutes is up, I will stop the timing and measure how much oxygen has been collected.

The apparatus and how to set it up.

List of apparatus:

• 1x Potato
• 1x Metal borer
• 1x Metal tile
• 1x 10ml syringe
• 1x Bung
• 1x Metal clamp
• 1x Large beaker
• 1x Heat mat
• 1x Kettle
• 1x Tripod
• 1x Boiling tube rack
• 1x Stop watch
• 1x Thermometer
• 1x Safety glasses
• 1x Weighing scales
• 5x Boiling tubes
• 5x Foam cups
• 5x Plastic holders

To set up the apparatus first place the foam cup inside the plastic holder, then place the boiling tube and its contents into the cup, put the rubber end of the bung in the top of the boiling tube and the other end in the large beaker underneath the syringe. Before putting the syringe in the beaker full of water check there is no trapped air inside the syringe and then place it in the water. Once in the water put the metal clamp on the rubber tube at the top of the syringe (see Fig 2, next page)

Fig 2

Risk assessment.

Hydrogen Peroxide is a risk, as it is irritant towards eyes. To make sure the Hydrogen Peroxide does not get in my eyes I will take care when using it and wear safety glasses.

Analysis

I collected the following data from the two Tests I did investigating the rate of reaction between catalase, the potato enzymes, and hydrogen peroxide, the substance being broken down into oxygen:

Test 1

Temperature                 Oxygen collected

                                    in 3 mins

3oc        1cm3

17oc        3cm3

25oc        3.5cm3

33oc        3.5cm3

50oc        2.5cm3

Test 2

Temperature                  Oxygen collected

                                     in 3 mins

3oc        0.5cm3

17oc        1cm3

25oc        6cm3

33oc        6cm3

50oc        7cm3

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In Test 1 the results show that the optimum temperature was reached at about 25oc-35oc, because at 50oc the rate of reaction dropped to 2.5cm3 per minute. However in Test 2 the results show that the rate of reaction is still increasing after 35oc, the fact that the rate of reaction is slowing down suggests the optimum temperature has almost been reached, and I believe if I had done another Test at 60oc, the rate of reaction would have been 5 or 6cm3 per 3 mins, 1 or 2cm3 less than what the rate of reaction was at 50oc.

           

I believe there is a pattern in the graph on the opposite page, it supports my first prediction, which stated; the enzymes will denature once the temperature rises over the optimum. The graph clearly shows the rate of reaction made a minor drop once the temperature exceeded 40oc-45oc. However my prediction is slightly undermined by the results because I predicted a slightly more sudden drop in the reaction time.

I believe I have gained these results, due to reactions taking place between the enzymes active site and the substrate, known as the collision theory. However at high temperatures the drop in reaction times suggests the enzymes start to denature, therefore damaging the active site and preventing any more reactions between the catalase and hydrogen peroxide to take place.

My results also agree with the Q10 theory. This theory states:

The rate of reaction at, xoc + 10oc ÷ the rate of reaction at xoc = 2

                                 

For example                           7oc + 10oc ÷ 7oc = 2.3

However this theory only works with temperatures below 40oc

Evaluation

I took every precaution to make sure my investigation was as fair as possible, however an anomaly that occurred during the Test was that the results recorded in Test 2 were all much higher than the results in Test 1, I believe the cause for this is that the bits of potato in Test 2 all weighed slightly more than the bits in Test 1, meaning there would be a slightly larger surface area meaning more collisions could occur between the active site and substrate. This theory could be plausible, because I used a different set of electric scales in both tests. To improve this anomaly I would make sure that next time I used the same scales throughout.

General problems that occurred during the experiment can be seen overleaf.

The experiment gave good enough results that were able to support my prediction and I believe they are just about reliable enough to give a useful trend.

To improve my investigation I would have used higher temperatures then I did in the final experiments, so I could get a better understanding of my results. I would have taken more time measuring out the ph7 and the H2O2 and I would have checked the tightness of the clamp before starting the reaction. I would have also chosen to use an electronic water bath and made sure the surface area was always the same. However most importantly I would have tried to get the potato the same weight every time.

Another way to improve the investigation is to put 3cm3 of pH7 and hydrogen peroxide in a test tube and let the investigation run for 5 minutes instead of three, this way the reaction should have finished by the time is up, giving a very accurate and reliable result.

I would of extended my investigation by letting the reaction go on for five minutes instead of three, my reason for stating this is; with only allowing the reaction to take place over the course of three minutes one does not know weather the reaction has completely finished or not.  I would have also chosen seven temperatures instead of five; this would give a larger spread of temperatures telling me more about the enzyme than before.

Although doing these two changes would mean spending longer over the investigation, I believe it would be better in the long run because the results would definitely be more accurate.

To extend my investigation even more, I would be very interested in incorporating a test to see how much the surface area of the potato can affect the rate of reaction and how much the results alter, because the more catalase that is exposed the greater the rate of reaction.

To do this I would cut the potato cylinder into halves, thirds and quarters, See fig 3. I would then proceed with the investigation as normal.

Fig 3

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Overall I believe I conducted a clear, safe and reliable experiment with very few minor mistakes. I am pleased with the way the experiment has gone and think I have gained a good understanding of enzymes and the way in which they react.