Practical write up 2
To Investigate the Effect of Varying pH on the Rate of a Reaction Catalysed by Trypsin
Aims
I hope to determine the effect of varying pH on the rate of reaction catalysed/controlled by the enzyme trypsin.
Method
1. Add 4cm³ of the 2% trypsin solution in to 6 test tubes labelled A-F
2. Add 4cm³ of the appropriate buffer solution to each. ( I will be testing pH 4,5,7,8,8.8 and 10)
3. Add 4cm³ of distilled water and 4cm³ of the appropriate buffer solution to 6 control test tubes labelled CA-CF to see if the pH alone will affect anything.
4. Place all test tubes in a water bath (40-45oC) and put the 1cm x 1cm squares of film in to them and start the stop clock.
5. From time to time, check if the squares have cleared and note down the times.
6. Repeat 3 times.
Results
pH buffer
Actual pH
Time for the film to clear (s)
Rate of reaction
Mean
4
4
>20 mins
4
4
>20 mins
0
4
4
>20 mins
5
5
602
1.66
5
5
602
1.66
1.67
5
5
600
1.7
7
7
1075
0.93
7
7
1083
0.92
0.92
7
7
1097
0.91
8
8
212
4.72
8
8
214
4.67
4.72
8
8
210
4.76
9
8.8
800
1.25
9
8.8
1000
1
1.08
9
8.8
1000
1
10
10
>20 mins
10
10
>20 mins
0
10
10
>20 mins
Conclusion
From the graph, I can conclude that enzymes can only function within a narrow pH range. They have an optimum pH that they function at, in this case around pH 8 and the further you deviate from that, the slower the rate of reaction because the enzyme functions less and less effectively. We can see from the graph that the enzyme stops functioning at pH 4 and 10 completely. This is because it has denatured. The pH affects hydrogen ions holding the secondary structure of the enzyme together and so changes the shape of the enzyme and its active site, making it impossible for the substrate to bind to form the enzyme substrate complex.
Evaluation
A result I did not expect was for pH 7. It should have been higher so that the graph had a smoother bell shape to it. This could be because the pH 7 test was done in a different water bath to where I was
Aims
I hope to determine the effect of varying pH on the rate of reaction catalysed/controlled by the enzyme trypsin.
Method
1. Add 4cm³ of the 2% trypsin solution in to 6 test tubes labelled A-F
2. Add 4cm³ of the appropriate buffer solution to each. ( I will be testing pH 4,5,7,8,8.8 and 10)
3. Add 4cm³ of distilled water and 4cm³ of the appropriate buffer solution to 6 control test tubes labelled CA-CF to see if the pH alone will affect anything.
4. Place all test tubes in a water bath (40-45oC) and put the 1cm x 1cm squares of film in to them and start the stop clock.
5. From time to time, check if the squares have cleared and note down the times.
6. Repeat 3 times.
Results
pH buffer
Actual pH
Time for the film to clear (s)
Rate of reaction
Mean
4
4
>20 mins
4
4
>20 mins
0
4
4
>20 mins
5
5
602
1.66
5
5
602
1.66
1.67
5
5
600
1.7
7
7
1075
0.93
7
7
1083
0.92
0.92
7
7
1097
0.91
8
8
212
4.72
8
8
214
4.67
4.72
8
8
210
4.76
9
8.8
800
1.25
9
8.8
1000
1
1.08
9
8.8
1000
1
10
10
>20 mins
10
10
>20 mins
0
10
10
>20 mins
Conclusion
From the graph, I can conclude that enzymes can only function within a narrow pH range. They have an optimum pH that they function at, in this case around pH 8 and the further you deviate from that, the slower the rate of reaction because the enzyme functions less and less effectively. We can see from the graph that the enzyme stops functioning at pH 4 and 10 completely. This is because it has denatured. The pH affects hydrogen ions holding the secondary structure of the enzyme together and so changes the shape of the enzyme and its active site, making it impossible for the substrate to bind to form the enzyme substrate complex.
Evaluation
A result I did not expect was for pH 7. It should have been higher so that the graph had a smoother bell shape to it. This could be because the pH 7 test was done in a different water bath to where I was