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The Effect of Temperature on Enzyme Activity

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The Effect of Temperature on Enzyme
Activity

Introduction

The success of living organisms is due to the cell's ability to make
protein catalysts, or enzymes, each with precisely specified
properties. Each enzyme has a unique shape containing an active site.
This active site binds a particular set of other molecules (called
substrates) in such a way as to speed up a particular one of the many
chemical reactions that the substrates can undergo (by a factor of up
to 1014). Like all other catalysts, enzyme molecules themselves are
not changed after participating in a reaction and therefore can
function over and over again.

Enzymes are usually irreversibly damaged (or denatured) under certain
conditions such as high temperatures above about 45oC. The pH affects
the activity of enzymes, in a similar way to temperature. A pH that's
much higher or lower than the optimum pH causes the enzyme to
denature. When a protein is denatured by heat or change in pH, the
folding of the enzyme and its active site is messed up and it does not
work.

Large food molecules must be broken down into smaller molecules before
our cells can use them. The stomach produces protease enzymes, which
break down large protein molecules into smaller ones. The stomach also
produces hydrochloric acid (HCl) to give the right pH for the protease
enzymes to work (pH2 - acidic). Protease enzymes in the small
intestine then break down the smaller proteins into amino acids.

In our experiment we will use will be pepsin, one of the protease
enzymes found in the stomach. We will use it to break down another
protein, albumen (egg white). The albumen is cloudy, but when we add
the pepsin at the right pH and temperature it will turn clear, meaning
that the pepsin enzyme has broken down the albumen into smaller
protein molecules. We will place a bit of paper with a cross drawn on
it under the bottom of the test tube, and time how long it takes for
the cross to become clearly visible.

Diagram

Preliminary work

I will carry out some preliminary work to find out the pH required for
optimal pepsin activity.

Variables:

Changed variables - pH (0.5, 1, 1.5, 2 molar HCl)

Controlled variables -Same equipment

-Same temperature (40ºc)

-Same person timing

-Same person judging when cross is clearly visible

-Same amount of albumen used in each experiment

-Same amount of pepsin used in each experiment

Measured variables -Time taken for the albumen solution to clear

Method:

1. Get out a thermostatically controlled water bath and set it to
40ºC.

2. Add 2 molar HCl to a test tube and place in water bath.

3. Add pepsin to the test tube.

4. Add the albumen. Start the stop watch when the albumen is added.

5. Time how long it takes to see the cross on the paper through the
bottom of the test tube (when the albumen changes from cloudy to
clear the reaction is complete).

6. Record the time in a result table. Repeat the experiment.

7. Repeat steps 2-6 using 0.5, 1 and 1.5 molar HCl (dilute 2 molar
HCl with water to make correct concentrations).

Results Table:

HCl concentration (molar)

Time (seconds)

Experiment 1

Experiment 2

0.5

102

106

1

72

69

1.5

58

55

2

45

43

Conclusion:

The results show that the rate of reaction is fastest using 2 molar
HCl. Using this information obtained from preliminary work I will use
2 molar HCl for my experiment measuring the effect of pH on the rate
of reaction.

Prediction

I predict that the rate of reaction will increase with increasing
temperature until the optimum temperature for the reaction is reached.
Above the optimum temperature the rate of reaction will fall to zero
very quickly, as the enzyme can not fold correctly and becomes
inactive. I also predict that the temperature for the optimal rate of
reaction for pepsin will be about 37oC as this is body temperature. As
I said in the Introduction enzymes I think at about 50ºC and above the
enzyme will be denatured and inactive. Below the optimum temperature
the reaction time will get increasingly slower for each lower
temperature.

Variables

Changed variables -Temperature of the water bath (10, 20, 30, 40, 50,
60, 70oC)

Controlled variables -Same equipment

-Same HCl concentration

-Same person timing

-Same person judging when cross is clearly visible

-Same amount of albumen used in each experiment

-Same amount of pepsin used in each experiment

Measured variables -Time taken for the albumen solution to clear

Method

1. Get out a thermostatically controlled water bath and set it to
20ºC.

2. Add 2 molar HCl to a test tube and place in water bath.

3. Add pepsin to the test tube.

4. Add the albumen. Start the stop watch when the albumen is added.

5. Time how long it takes to see the cross on the paper through the
bottom of the test tube (when the albumen changes from cloudy to
clear the reaction is complete).

6. Record the time in a result table. Repeat the experiment two more
times.

7. Reset the temperature of the water bath 30, 40, 50, 60 and 70oC
and repeat steps 2-6 at each temperature.

Results

Temperature of Pepsin-Albumen solution (oC)

Time taken to clearly see cross(seconds)

Experiment1

Experiment2

Experiment3

Average

10

180

180

210

190

20

170

80

95

115

30

64

67

65

65

40

56

45

43

48

50

32

36

34

34

60

105

108

107

107

70

220

217

210

216

Conclusion

The results show that the rate of reaction increased with increasing
temperature up to 50oC. Above 50oC the rate of reaction then drops
quickly. Therefore in our experiment 50oC is about the optimum rate of
reaction for pepsin.

The results support my prediction to an extent but, my prediction was
wrong about what I thought the temperature for the optimum rate of
reaction would be. I thought the rate of reaction of pepsin would be
fastest at about 37ºC because this is body temperature. However the
rate of reaction increased as the temperature increased up to about
50ºc. Above the optimum temperature the rate of reaction fell to zero
very quickly, as the enzyme denatured. I thought pepsin would denature
at 50ºc because as I said in the Introduction enzymes usually denature
around 45oC, but in fact it started to denature at 60ºc.

Evaluation

The procedure we used wasn't too bad, but it could have been better. I
don't think looking at a cross drawn on paper and put underneath the
test tube containing the albumen and pepsin solution is a very
accurate way of telling when the reaction is finished. This is because
the human eye is not very accurate. This is why I think we got an
anomalous result: our first attempt at 20ºC was 170 seconds whereas
the other two were 80 and 95 seconds. I think this is because he must
have called out for the stop watch to be stopped too late. The repeat
attempts were mostly strong and good apart from this one anomalous
result. If I were to do the experiment again I would use a
spectrophotometer to improve the quality of the results. A
spectrophotometer is a machine which measures the optical density of
solutions and would have accurately recorded the time taken for the
albumen to clear.

Also because the test tube had to be taken out of the water bath to
put it over the cross on the paper the temperature during each
experiment would have changed. It may have been because of this that
the temperature recorded for the optimum rate of reaction was 50oC.
Although the experiment may have started at this temperature it would
have dropped when the test tube was taken out of the water bath. The
experiment could therefore be improved by finding a way of keeping the
temperature constant while monitoring the reaction.

I compared my results to a friend's in my class who also did the
experiment.

Temperature of Pepsin-Albumen solution (oC)

Time taken to clearly see cross (seconds)

Average of my experiments

Average of my friend's experiments

10

190

201

20

115

120

30

65

73

40

48

50

50

34

35

60

107

110

70

216

205

My friend's results were similar to my results. He also found that the
rate of reaction was optimum at about 50oC. All of the secondary
source's results are slightly higher except the one at 70oC. I think
this is because they must have had someone looking at the cross who
called late, or we had someone calling early. Now I have put my
results against a secondary source I think that my results are quite
reliable because they are pretty much the same. The pattern is very
similar.

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