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The purpose of this investigation was to test how the enzyme catalase is affected by changes in pH and temperature.  This was done by calculating the rate at which oxygen was produced when catalase acted on hydrogen peroxide in pH buffer solutions of pH 3, 4, 5, 6, 7 and 8 and at temperatures of 10, 20, 30, 40, 50 and 60oC.

Abstract and Introduction

Abstract

The purpose of this investigation was to test how the enzyme catalase is affected by changes in pH and temperature.  This was done by calculating the rate at which oxygen was produced when catalase acted on hydrogen peroxide in pH buffer solutions of pH 3, 4, 5, 6, 7 and 8 and at temperatures of 10, 20, 30, 40, 50 and 60oC.

A t-test was carried out to test the significance of the results.  The hypothesis that the rate of reaction of catalase will be significantly affected by pH and temperature changes could not be fully accepted as the statistical test showed that there was a 50% possibility that the changes in rate of reaction when the temperature was altered were due to chance.  The results gained when the pH value was altered were statistically significant.


Introduction

Hypothesis:

The rate of reaction of catalase will be significantly affected by pH and temperature changes.

The enzyme catalase is present in all aerobic tissues and catalyses the breakdown of hydrogen peroxide into water and oxygen. Hydrogen peroxide is a toxic by-product of many bio-chemical reactions within organisms, including aerobic respiration.

Michaelis and Menton (1913) described the change in the rate of reaction of an enzyme-catalysed reaction when the concentration of substrate is changed.  This led to many rate studies on enzymes being carried out in which it was found that as well as changes in enzyme and substrate concentration, “an enzyme’s catalytic activity is strongly affected by temperature and pH conditions” (Michaelis, 1913).

I have chosen to carry out an experiment on catalase to see the extent to which the way pH and temperature changes affect an enzyme’s rate of reaction can be applied to catalase.  I have not been able to find any reports on similar work with catalase but I predict that it will act in a way similar to that of the reaction of other enzymes to pH and temperature changes.

I predict that an increase in temperature will increase the kinetic energy of the substrate and enzyme molecules.  This will cause them to collide more often with each other which will increase the rate of reaction.  The reaction will support the Q10 rule, i.e. a 10oC rise in temperature will result a doubling of the reaction rate.  This will only happen up to about 40oC because a rise in temperature causes the atoms making up enzyme molecules vibrate.  This will break the hydrogen bonds holding the catalase molecules in their precise three-dimensional shape and, therefore, denature the enzyme so that the hydrogen peroxide molecules cannot fit the active sites.  Catalase will have an optimum temperature of about 40oC when denaturation will begin to have an affect and the rate of reaction will begin to decrease.

I predict that catalase will also have an optimum pH.  This is because the hydrogen bonds holding an enzyme in its precise three-dimensional molecular shape can be broken by the concentration of hydrogen ions present, reducing its ability to form enzyme-substrate complexes.  pH is a measure of hydrogen ion concentration and a change of one pH point represents a tenfold change in the hydrogen ion concentration.  Catalase will, therefore, become denatured when the pH deviates from the optimum due to the breaking of the hydrogen bonds.

The independent variables in this experiment will be the temperatures and pH levels at which the catalase will be tested.  The temperatures it is tested at will be 10oC, 20oC, 30oC, 40oC, 50oC and 60oC.  The pH levels it is tested at will be pH 3, 4, 5, 6, 7 and 8.

The dependent variable in this experiment will be the time it takes for the reaction to produce 5cm of oxygen and, therefore, the rate of the reaction.

The method that will be used for the investigation is outlined below.

1)  Cut cylinder of potato tuber tissue (6cm long) with cork borer.  Cut 60, 1mm thick discs and place under water in a Petri dish.

2)  Set up apparatus.

3)  Remove bung from boiling tube.  Place 5cm3 of buffer solution at pH 3 into boiling tube with a syringe.  Add 10 potato discs.  Add 5cm3 of hydrogen peroxide with another syringe.

4)  Replace the bung.  Start stop clock.

5)  Note how long it takes for the fluid to rise through 5cm.

6)  Open the clip.  Repeat experiment twice more.

7)  Remove bung and thoroughly wash out boiling tube.

8)  Follow same procedure with pH 4, 5, 6 ,7 and 8 in turn.  Each time use a new set of potato discs and a clean syringe.

9)  Cut another cylinder of potato tuber tissue and slice it into 60, 1mm thick discs.  Place them under water.

10)  Remove bung from boiling tube.  Place 5cm3 of water in boiling tube with a syringe.  Add 10 potato discs.

11)  Place boiling tube in water bath at 10oC.  Add 5cm3 of hydrogen peroxide.

12)  Replace the bung and close the clip.  Start stop clock.

13)  Note how long it takes for fluid to rise through 5cm.  Open clip.  Repeat experiment twice more.

14)  Wash out boiling tube and repeat step 10).

15)  Heat water to 20oC.  Add 5cm3 of hydrogen peroxide.

16)  Repeat steps 11) - 13).

17)  Repeat same procedure at 30oC, 40oC, 50oC and 60oC.


Method

Apparatus

Razor blade,
Cork borer,
Ruler (mm units),
Ceramic tile,
Boiling tube (with bored rubber bung),
Stand, boss, clamp,
Manometer tube (3mm diameter),
Glass beaker,
Thermometer (0-100oC),
Syringes (5cm3) x 8,
Clip,
Stop clock,
Petri dish,
Bunsen burner,
Mat, tripod, gauze,
Wax pencil,
Potato tuber,
Manometer fluid,
Ice,
Hydrogen peroxide,
pH 3, 4, 5, 6, 7, and 8 buffer solutions. 


Method

The catalase enzyme in a potato was used for this investigation.  A cork borer was used to cut a cylinder of potato tuber tissue.  The cylinder was placed on a ceramic tile and a razor blade was used to cut it to 6cm in length.  The razor blade was then used to slice the cylinder into sixty, 1mm thick discs.  The thickness of the discs were measured as accurately as possible by placing the cylinder against a ruler with millimetre units marked on.  As the discs were cut they were placed under water in a Petri dish.  The apparatus were set up as shown above with the clip open.  The stand, clamp and boss were used to hold the boiling tube.  A wax pencil was used to mark on the right hand manometer tube the level of the fluid and another mark was made 5cm up from the first.  The bung was then removed from the boiling tube.  A syringe was used to place 5cm3 of buffer solution at pH 3 into the boiling tube.  Care was taken to count ten potato discs and add them to the boiling tube.  A clean syringe was used to add 5cm3 of hydrogen peroxide.  A clean syringe was used so that the buffer solution and hydrogen peroxide were not mixed and further experiments were not altered which kept the experiment fairer.  The bung was fitted to the boiling tube immediately after the hydrogen peroxide was added, making sure that it was air tight.  This ensured that no oxygen produced during the reaction was lost which would alter the results.  The stop clock was started.  The boiling tube was gently agitated to increase the collisions between the catalase and hydrogen peroxide molecules and the clock was stopped when the manometer fluid had risen through the 5cm marked on the tube and the time was recorded.  The clip at the top of the boiling tube was opened so the fluid fell back to its original position.  Once level with the lowest mark on the tube, the clip was closed and the stop clock was started.  The time taken for the fluid to rise through 5cm was again recorded.  The clip was opened and the procedure was carried out once more.  After the third reading had been taken, the clip was opened and the bung was removed.  The boiling tube was then washed out thoroughly so that no pH 3 buffer solution or hydrogen peroxide was left behind which could alter the results of the experiment when different substances are being tested.  The whole experiment was then repeated with buffer solutions of pH 4, 5, 6, 7, and 8 in turn.  A new set of ten potato discs and a clean syringe was used for each pH buffer solution so that the results were not affected.  The whole experiment to test the affect of pH on catalase activity was carried out at room temperature. 

To test the affect of temperature the boiling tube was washed out and a clean syringe was used to place 5cm3 of water in it.  Another sixty, 1mm thick potato discs were cut as before and ten discs were added to the boiling tube.  The boiling tube was placed in a water bath and ice was used to decrease the temperature to 10oC.  The potato discs were left a couple of minutes to adjust to the temperature.  A clean syringe was used to add 5cm3 of hydrogen peroxide.  The bung was then replaced, the clip was closed and the stop clock was started.  The boiling tube was gently agitated and the clock was stopped when the manometer fluid had risen through the 5cm and the time was recorded.  The clip was opened to allow the fluid to return to the original mark.  The clip was closed and the procedure repeated twice more.  The clip was opened again and the bung was removed.  The boiling tube was washed out, another 5cm3 of water was placed in it and a new set of ten potato discs were added.  The boiling tube was placed in the water bath and a Bunsen burner was used to heat the water bath to 20oC.  Again, the potato discs were left to adjust to the temperature for a couple of minutes before the hydrogen peroxide was added.  5cm3 of hydrogen peroxide was added, the bung was replaced and the clip was closed.  The stop clock was started and the time was recorded when the fluid had risen 5cm.  Two more readings were taken at 20oC.  The experiment was the repeated at 30oC, 40oC, 50oC and 60oC.  For each new temperature a new set of ten potato discs were used.

Every reading throughout the experiments was recorded three times so that an average could be calculated.


Results

Results

The time taken for the reaction to produce 5cm of oxygen was noted in seconds. The rate of reaction was then calculated by dividing 1000 by the time taken. The rate of reaction is expressed in s-1.

Table Showing The Effect Of pH Changes

pH
Time taken to produce 5cm of oxygen (seconds)
Rate of reaction (s-1)
3
194.4
5.14
3
240.6
4.16
3
235.2
4.25
Average
223.4
4.52
4
128.4
7.79
4
91.8
10.89
4
150.6
6.64
Average
123.6
8.44
5
28.8
34.72
5
34.8
28.74
5
60.6
16.50
Average
41.4
26.65
6
30
33.33
6
25.2
39.68
6
29.4
34.01
Average
28.2
35.67
7
26.4
37.88
7
23.4
42.74
7
24
41.67
Average
24.6
40.76
8
29.4
34.01
8
29.4
34.01
8
34.2
29.24
Average
31
32.42

Table Showing Effect Of Temperature Changes

Temp(°C)
Time taken to produce 5cm of oxygen (seconds)
Rate of reaction(s-1)
10
63
15.87
10
120.6
8.29
10
144
6.94
Average
109.4
10.37
20
30
33.33
20
80.4
12.44
20
130.2
7.68
Average
80.2
17.82
30
63.6
15.72
30
69
14.49
30
70.8
14.12
Average
67.8
14.78
40
24.6
40.65
40
64.2
15.58
40
73.2
13.66
Average
54
23.30
50
27.6
36.23
50
68.4
14.62
50
76.2
13.12
Average
57.4
21.32
60
0
-
60
0
-
60
0
-
Average
0
-

Discussion

Discussion

The graphs and results tables show that catalase has both an optimum pH and temperature. The rate of reaction increased as the pH was increased until pH 7. The rate of reaction decreased again in the test with the buffer solution at pH 8. This shows that catalase works best in conditions where the hydrogen ion concentration is at the neutral point.

The rate of reaction also increased up to 40oC due to the increased kinetic energy of the catalase and hydrogen peroxide molecules. 40oC was the optimum temperature for catalase. When the temperature was increased to 50oC the rate of reaction decreased as the catalase molecules began to denature and at 60oC the enzyme was fully denatured and the reaction stopped.

The graph showing the effect of temperature on the rate of reaction does show a couple of anomalous results. The rate of reaction increases quite rapidly between 10oC and 20oC but then decreases when the temperature is increased to 30oC. It is still possible, however, to see the general trend of an increasing rate of reaction. These anomalies could have been obtained because of limitations of the techniques used which caused inaccuracies.

A limitation which could have caused inaccuracies in the results was that there could have been slight variations in the thickness of the potato discs. Although care was taken to slice the potato cylinder into 1mm thick discs by using a razor blade and a millimetre ruler, it is difficult to cut all discs to exactly the same thickness. If this experiment was carried out again it would be more accurate to mince the potato tuber and use the same mass of potato each time.

The investigation could be further improved by using closer pH and temperature differences. For example, testing reaction rate at pH 3, 3.5, 4, 4.5 etc and at 10oC, 15oC, 20oC, 25oC etc. This would mean that an optimum pH and temperature could have been estimated more accurately. Although the graphs show an optimum of pH 7 and 40oC, the optimums could actually be somewhere between pH 7.5 and 35oC for example.

Enzymes are an essential part of both plant and animal life. They are catalysts for metabolic reactions within organisms and without them the reactions would happen too slowly to sustain the life of the organism. Without catalase, hydrogen peroxide would build up faster than it could be broken down and its toxic properties would be lethal. Without the enzymes involved in respiration an organism would not be able to respire at a rate fast enough to survive.

This investigation has shown that both temperature and pH play an important part in the activity of an enzyme. It is, therefore, necessary for these two factors to be kept fairly constant in order for enzymes to be most effective. Endothermic animals use metabolic activities to keep their body temperature constant which is usually within the range of 35-44oC. Exothermic animals use behavioural methods to regulate their body temperature. The temperature of soil is important to plant species as germination and growth depend on a suitable temperature. The activity of soil organisms is affected by changes in temperature, for example, earthworms become dormant at low temperatures.

The pH of a soil influences the availability of certain minerals to plants. The results of this investigation suggest that a potato will benefit the best from a soil of about pH 7 and temperature of about 40oC. Very high or low pH values inhibit the growth and functioning of nearly all roots due to the influence on enzyme activity, affecting the rest of the plant.

pH values within animals must also be kept constant. For example, mammalian blood must be kept at pH 7.4 and any serious deviation from this is fatal. Animals possessing a body fluid regulate their internal pH at a constant value by the buffering action of substances present in the body fluids and by hormones.