Investigating the effect of hydrogenperoxide on catalase if you change the temperature.

The effect of hydrogen peroxide on catalase if you change the temperature

AIM
Effect of temperature of the action of the Enzyme Catalase.

PLANNING

Background Knowledge

An enzyme is a biological catalyst, it alter the rate of reaction without being changed itself. Enzymes are proteins; they have a very precise three-dimensional shape, which forms a one specific active site on the enzyme. Each enzyme can only convert one kind of substrate molecule in to one kind of product molecule. These are specific.

What affects Enzymes?

· Temperature- Enzymes stop working if the temperature rises above 40ºC. Increasing the temperature alters the 3D shape and so the enzyme can no longer fit the substrate.

· pH- They work best in neutral conditions neither acidic nor alkaline.

What affect does catalase have?

Catalase is a very fast reacting enzyme, it is found in many living cells, it breaks down hydrogen peroxide to water and oxygen. In fact one molecule of it can deal with six million molecules of hydrogen peroxide in 1 minute. Hydrogen peroxide is toxic so needs to be changed into harmless substances.

Catalase
Hydrogen peroxide water + oxygen
2H2O2 2H2O + O2

References to practicals referring to enzymes

· Biology for You Pg 30 - Experiment 3.1

From looking at this I found out that catalase reacts with hydrogen peroxide to give out water and oxygen. Oxygen bubbles produce froth on the surface of the solution. In my forthcoming experiment I will expect to see froth being produced.

· Biology- Nelson Science Pg 25 - Picture 4

From looking at this graph, see below. I have learnt that the affect of temperature does in fact change the rate of reaction. From the graph the reaction reaches 40ºC but then denatures and the rate of the reaction decreases. The rate falls rapidly suggesting denaturing.
Taking this information into account I would expect the enzyme catalase to show a similar pattern with respect to the temperature.

In order to observe the effect of temperature on catalase we will be maintaining in the amount of oxygen released. The oxygen produces a froth which we will then measure in mm and the volume of oxygen given off which will be measure in cm³

Method- measuring the height of froth and volume of oxygen

1. Put work shirt on and goggles on. Carry out the rest of safety precautions.
2. Gather equipment as shown on diagram1.
3. Using a cork borer make 5 cylinders from the large potato.
4. Cut them into all the same length (6cm)
5. Using a pestle and mortar mash up each cylinder separately.
6. Measure 25ml of hydrogen peroxide using a measuring cylinder.
7. Select the temperature you are going to study

0ºC- iced water
25ºC-no extra equipment
37ºC-water bath required
55ºC-water bath required
100ºC-beaker of boiling water

8. Place on mashed cylinder into a boiling tube add the measured hydrogen peroxide and attach the rubber bung connected to the measuring syringe.
9. Start stop watch and record volume of gas collected every 30 seconds. At the same time measure the amount of froth produced at 30 seconds intervals

Apparatus

· 5 beakers
· 5 test tubes
· Thermometers
· Cork borer
· Potato
· Ruler
· Knife
· Tile
· Measuring syringe
· Heat proof mat
· Bunsen burner
· Tri-pod
· Wire gauze
· Pestle and mortar
· Hydrogen peroxide
· Matches
· Spills
· Ice cubes
· Water bath
· Goggles
· Spatula
· Stopwatch
· Measuring cylinder

Fair test

In this investigation I will keep constant the following

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Apparatus

Fair test

In this investigation I will keep constant the following

· The surface area of the potato. I will use the mashed up form as it will be a faster reaction as there is more area to react on, as we have to consider the time span.
· The same volume of hydrogen peroxide in each part of the investigation.
· The same size equipment e.g. boiling tubes as the readings for the results will be wrong if this is not constant.
· Use the same method for each experiment so that there won´t be any major differences. Only alter the temperature.
· Keep the amount of potato the same amount.
· Measure the temperature with a thermometer.

Accuracy

In order to make my investigation go to plan I will be as accurate as I can be so I will measure to the correct measuring size.

· Measure the volume in cm³ and amount of potato in grams to make sure that they are exactly the same mass before using them in the experiment.
· Do the experiment three times to ensure that there isn´t an odd result. Three is a good number to use as you can see if there is one odd one where if you just done the experiment twice then you wouldn´t know which one odd and which isn´t.
· Also to average out the results.

Safety precautions

· Wear goggles
· Tuck tie in skirt
· Wear work shirt
· Handle the hydrogen peroxide with care as it is corrosive and an irritant

Predictions and Reasons

From my research I think that the enzymes will denature after 40ºC and any other temperature above that. Reason being that enzymes are proteins and their structure is three-dimensional. Increasing the temperature disturbs the intra molecular bonds that hold the 3D shape. Because of this the shape is altered. Enzymes have an active site. This fits into the substrate molecular (see diagram2-lock and key). If the active site is altered the substrate will no longer fit in and so the enzyme doesn´t work properly.
The rise of reaction rate is also due to the increase in temperature, relating to the kinetic theory. The higher the temperature, the faster they move. This happens but only to an optimum of 40ºC. The curve leading up to the optimum point is gradual but as it is reached it falls dramatically. The reason being that the active site is destroyed therefore no reaction can take place as there is only one specific active site per substrate.

OBTAINING EVIDENCE

Below are my table of results which show the height of froth produced in cm and the volume of oxygen in cm³ for each of the three tests at each of the five temperatures studied.
TEMPERATURE: 10ºC
TEST 1 TEST2 2 TEST 3

TIME (mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 3 3 2.4 9 2 4
1 3.7 6 3 10 3 8
1.5 4.2 8 3.3 11 4.3 12
2 4.8 10 3.5 12 5.4 12
2.5 5.3 11 3.9 13 6 12
3 5.7 12 4 13 6.2 13
3.5 6.5 12 4.2 13 7.4 13
4 6.8 13 4.4 13 8 14
4.5 7.5 13 4.4 13 8 14
5 8.2 13 4.4 13 8 14

TEMPERATURE: 25ºC

TEST 1 TEST2 2 TEST 3

TIME(mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 3 9 4 5 3 8
1 5 14 6 10 4.9 12
1.5 6 18 6.5 14 5.8 15
2 7.5 20 7 18 7.6 19
2.5 9 20 8 20 8.2 20
3 10 20 9 21 9.1 21
3.5 10 20 9 21 10 22
4 10 20 9 21 10 22
4.5 10 20 9 21 10 22
5 10 20 9 21 10 22

TEMPERATURE: 37ºC

TEST 1 TEST2 2 TEST 3

TIME(mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 4 7 5 12 4.5 10
1 5.5 14 8 20 6 16
1.5 7 19 10 26 8 22
2 9 22 11 28 10 26
2.5 10 28 12 30 11 28
3 10 28 12 30 11 28
3.5 10 28 12 30 11 28
4 10 28 12 30 11 28
4.5 10 28 12 30 11 28
5 10 28 12 30 11 28

TEMPERATURE: 55ºC
TEST 1 TEST2 2 TEST 3

TIME(mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 4 12 5 14 6 15
1 6 18 6 19 7 20
1.5 7 22 6.5 22 8 22
2 8 24 8 24 8 24
2.5 8 25 8 25 8 25
3 8 26 8 25 8 26
3.5 8 26 8 26 8 26
4 8 26 8 26 8 26
4.5 8 26 8 26 8 26
5 8 26 8 26 8 26

TEMPERATURE : 100ºC
TEST 1 TEST2 2 TEST 3

TIME(mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 0.1 0.5 0.1 1 0.1 1
1 0.1 0.5 0.1 1 0.1 1
1.5 0.1 0.5 0.1 1 0.1 1
2 0.1 0.5 0.1 1 0.1 1
2.5 0.1 0.5 0.1 1 0.1 1
3 0.1 0.5 0.1 1 0.1 1
3.5 0.1 0.5 0.1 1 0.1 1
4 0.1 0.5 0.1 1 0.1 1
4.5 0.1 0.5 0.1 1 0.1 1
5 0.1 0.5 0.1 1 0.1 1

AVERAGES
Table of averages from each of the above temperatures

TEMPERATURE: 10ºC

TIME(mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 2.5 5.0
1 3.2 8.0
1.5 3.9 10.3
2 4.6 11.3
2.5 5.1 12.0
3 5.3 12.7
3.5 6.0 12.7
4 6.4 13.3
4.5 6.6 13.3
5 6.9 13.3

TEMPERATURE: 25ºC

TIME(mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 3.3 7.3
1 5.3 12.0
1.5 6.1 15.7
2 7.4 19.0
2.5 8.4 20.0
3 9.4 20.6
3.5 9.7 21.0
4 9.7 21.0
4.5 9.7 21.0
5 9.7 21.0

TEMPERATURE: 37ºC

TIME(mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 4.5 9.7
1 6.5 16.6
1.5 8.5 22.3
2 10 25.3
2.5 10 28.7
3 10 28.7
3.5 10 28.7
4 10 28.7
4.5 10 28.7
5 10 28.7

TEMPERATURE: 55ºC

TIME (mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 5.0 13.7
1 6.3 19.0
1.5 7.1 22.0
2 8.0 24.0
2.5 8.0 25.0
3 8.0 25.7
3.5 8.0 26.0
4 8.0 26.0
4.5 8.0 26.0
5 8.0 26.0

TEMPERATURE: 100ºC

TIME (mins) HEIGHT OF FROTHcm VOLUMEOF OXYGEN(cm³)
0.5 0.1 0.83
1 0.1 0.83
1.5 0.1 0.83
2 0.1 0.83
2.5 0.1 0.83
3 0.1 0.83
3.5 0.1 0.83
4 0.1 0.83
4.5 0.1 0.83
5 0.1 0.83

These two tables show the average measurement that we recorded for each temperature.

HEIGHT

Time (mins)
Temperature (ºC) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
10ºC 0 2.5 3.2 3.9 4.6 5.0 5.3 6.0 6.4 6.6 6.9
25ºC 0 3.3 5.3 6.1 7.4 8.4 9.4 9.4 9.4 9.4 9.4
37ºC 0 4.5 6.5 8.5 10 10 10 10 10 10 10
55ºC 0 5.0 6.3 7.1 8 8 8 8 8 8 8
100ºC 0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

VOLUME

Time (mins)
Temperature (ºC) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
10ºC 0 5.3 8 10.3 11.3 12 12.7 12.7 13.3 13.3 13.3
25ºC 0 7.3 12 26.7 19 20 20.6 21 21 21 21
37ºC 0 9.7 16.6 22.3 25.3 26.7 26.7 26.7 26.7 26.7 26.7
55ºC 0 13.7 19 22 24 25 26.7 26 26 26 26
100ºC 0 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8

Analysing results and Conclusion

From my results it appears that catalase works best at 37ºC, and it is virtually denatured at boiling point.
Looking at the initial part of the reaction (see graph 1) it is clear that the gradient at the beginning gets steeper when looking at the temperatures between 10ºC-55ºC. At each temperature the line levels off towards the end of five minutes. Looking at graph 2, there is a steady rise in height of froth up to 37ºC and then a gradual fall up to 100ºC.
Looking at my background knowledge and prior experiments using enzymes I can explain my results as follows.
Kinetic theory states that particles, which gain heat energy, move more quickly. In our case the reacting particles are the substrate (hydrogen peroxide) and the enzyme catalase. As the temperature is increased the particles of hydrogen peroxide have more energy therefore they collide with the potato more frequently and so increasing the rate at which the product is formed. However at a certain temperature this is no longer the case. This is because enzymes are proteins and proteins can be denatured at high temperatures. This is because proteins have a 3D shape. In our case the catalase has a certain shape that the substrate fits into. At high temperatures the active site on the enzyme is altered, see diagram below.

(Diagram showing active site on the enzyme is altered therefore stopping products being formed)

This stops the substrate from 'fitting´ and so no product is formed.
My results do not totally support or undermine my original prediction. The reason being that on graph 1, my results suit my prediction. It shows that the temperature, 37ºC was the fastest and 100ºC is when the enzyme denatures. But in graph 2, my results undermine my original prediction as at 55ºC the reaction still takes place where as in my prediction I stated that enzymes would denature at 40ºC approximately, I didn´t expect this is happen.

Evaluation

In my investigation I was pleased with my achievements.
In my method, keeping the temperature constant throughout the investigation was hard to maintain, as the temperature of the contents of the tube would change quite quickly and therefore the hydrogen peroxide wouldn´t be at the temperature required. To overcome this problem I could keep the test tubes in a hot water bath for all the temperatures making sure that the water bath was the suitable depth. This would ensure constant temperature throughout the whole 5 mins. Also another problem that I encountered was to keep the height of the froth fair. I measured the height of the froth with a 30cm ruler against the test tube rack, with the support of my hand. As I was measuring, my hand would move from time to time and therefore didn´t know where I should place my ruler afterwards. To over come this I should attach the ruler onto the test tube rack with cello tape, as it is transparent or maybe use a pointer.
With respect to I measured the height of froth in cm, but to be more precise I should have measured it in mm. To over come this I should use a ruler with mm readings. Also another problem that I observed on accuracy was that I didn´t allow the temperature to equilibrate to the right temperature. In this case I wasn´t using the correct temperature that I wanted, this could have led to some anomalous results. Ideally I should have brought the temperature of the hydrogen peroxide up to the needed temperature before adding to the potato.
Looking back at my results I found some anomalous results in my findings. When averaging I used these results, which could of made the average either lower or higher than it should be. To improve this I should have missed these results. Not including some sets of results when making averages may have led to better values.
My results are in line with those I predicted. Graphs indicate rise in temperature up a point leads to an increase in oxygen production. This is in line with kinetic theory. However it is very clear that after a certain temperature is reached the enzyme actually virtually stops. This supports my theory of lock and key fit.
However optimum activity of enzyme is at about 37ºC this is as we expected. But at 55ºC the enzyme is still not denatured according to my results. This is a higher temperature than I would expect. Possible not allowing solutions to reach temperatures selected has led to an inaccuracy. It may be that in fact that many temperatures of solutions were lower than we stated.
Overall, due to reliable repeats and in general predictions being confirmed I feel my results are reliable enough to make a conclusion.
The obvious thing I would improve about the measurements I made would be to increase the range of temperatures used. Especially between 55ºC-100ºC. In this way it may be clearer at the temperature which denaturing took place, and would possibly give a graph that resembled the graph in background knowledge.
Another way of improving this investigation is to change the method. I measure the volume of oxygen that was produced. In order to get pure oxygen without any other gases that are in the air I would use the same equipment but make sure that the gap between the rubber bung and solution was free from any other gases.