Maryam Jamshidi

Source:

http://mycourses.mycandi.ac.uk/pluginfile.php/237165
(Core practical)
/mod_resource/content/0/01_core_practicals/Activity_
2.11_Enzyme_concentration/Notes%20for%20writing
%20up%20Enzyme%20core%20practical%20_v1.pdf

Activity - Enzyme concentration and Enzyme activity

Purpose: to investigate the effect of the Trypsin concentration on the

rate of reaction on breaking down Casein, the protein found commonly in
milk.
Introduction
Enzymes are proteins that speed up particular reactions within cells. They
are biological catalysts as they take part in a reaction yet remain
unchanged at the end of the reaction, and although they have an impact
on the rate, they do not affect other components within the reaction. Most
catalysts are not selective as they catalyse more than one reaction.
Enzymes however, are highly selective and catalyse one specific reaction
only. This is due to the tertiary structure of the protein which generates
the specific shape of an enzyme molecule. A lot of enzymes consist of a
non-protein (cofactor) and a protein (globular) component. Inappropriate
temperatures and pH are able to denature proteins, which means the
polypeptide chains can be impacted, which can lead to a change in shape,
causing denaturing to take place for the enzyme. The optimum conditions
for an enzyme to work are those conditions that allow it to work at its
fastest rate. Different enzymes have different optimum temperatures and
pH levels, therefore, the catalytic activity within enzymes are pH and
temperature sensitive.
How enzymes work
For two molecules to react they must collide with one another in the right
direction (orientation) and with sufficient energy. Sufficient energy means
that between them they have enough energy to overcome the energy
barrier to reaction.
Lock and key theory:
In this theory, the substrate fits into the active site of the enzyme to form
an enzyme-substrate complex. Bonds break while the substrate is locked
in the enzyme-substrate complex, which will allow the product to be
produced.
Induced fit theory:
In this theory, the enzyme molecule changes shape as the substrate
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Maryam Jamshidi

(Core practical)

molecules gets close. The change in shape is 'induced' by the approaching

substrate molecule. This model relies on the fact that molecules are
flexible because single covalent bonds are free to rotate.
The product of these theories is a broken down version of the substrate, in
other words, when bonds break within a molecule, the molecule breaks
apart to even smaller molecules, where it will be able to be absorbed into
cells.
Hypothesis: The higher the concentration of enzyme, the faster the rate
of the reaction of the trypsin breaking down the casein.
Variables:
The independent variable of this experiment is the concentration of the
trypsin enzyme as water was added to dilute the enzyme concentration in
controlled amounts. The dependent variable of this experiment is the time
taken for the solution to reach 50%T in the colorimeter. The control
variables of the experiment are the types of enzymes used (the trypsin
and casein), the volume of the solution in the test tubes as it was always
at 5cm, and the volume of milk put into the solution was always at 3cm.
Method:
Apparatus:

Beaker of casein solution

Beaker of trypsin solution
Beaker of distilled water at pH9
6 x small glass Test tubes,
Test tube rack
Stop clock
2 x 5 cm3 syringe
1 cm3 syringe
Colorimeter

I started off by preparing my apparatus. I filled my first test tube with

5cm of the trypsin solution, the second with 4cm of the trypsin solution
and 1cm of water, the third with 3cm of trypsin solution and 2cm water,
and so on until no volume of trypsin was used in any more test tubes. I
then made a solution of 5cm of trypsin, 3cm of water, which is what I
used as a reference for my colorimeter. I then labelled each test tube so I
wouldnt mix them up during the experiment and so it would be easy to
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Maryam Jamshidi

(Core practical)

identify the different concentrations throughout the experiment. I turned

on the colorimeter and put in the reference test tube as I pressed the R
button. I then took it out, and put one of the test tubes into the
colorimeter. I pressed the graph button, then put 3cm of the milk solution
into the test tube, and pressed %T until it reached the reading of 50%T. I
began the stop clock as soon as I put the milk into the test tubes to be
able to see the rate of reaction in seconds. I did this for all concentrations,
including the solution without the trypsin enzyme in it.
Results and Conclusion:
rate of reaction
(1/t) /s-1

time to 50%T / s
Enzyme
conc. / gdm3

1
2
3
4
5

1
1
8
1
2
6
4.
5
3.
5

2
23
13.
3
12.
2
9.1
8.7
8

12 /
4.
2 /
4. 10.
1
4
3.
5 5.3
3.
2 6.5

5
17.
9
13.
2
8.8
1
7.7
6
6.0
6

6
15

7
1
3

8
1
7
1
3

1.5

3
2.5
7

9
24.3
5
18.7
6
14.8
8
10.1
7

5.9

mea
n

S
D

18

11

mea
n
0.05
7
0.09
1
0.12
1
0.16
0
0.21
2

SD
0.014
3
0.040
3
0.064
1
0.062
8
0.106
5

Maryam Jamshidi

(Core practical)

Effect Of Trypsin Concentration On Rate Of Reaction

0.250

0.200

0.150
rate of reaction/s-1

0.100

0.050

0.000
0.5

1.5

2.5

3.5

4.5

5.5

Enzyme concnetration / gdm-3

Through my class results I can see that as less water is used to dilute the
trypsin solution (so the stronger the concentration of the trypsin) the less
time it takes for the trypsin to break down the casein molecules in the
milk mixture added to the enzyme solution.
Interpretation:
It seems that the higher the trypsin concentration, will mean that there is more
trypsin enzymes within the given volume, which will allow the casein to hit an
active site of an enzyme more likely than a diluted solution as there are more
trypsin molecules available compared to water molecules.

Evaluation:
According to the standard deviation, there appears to be a very wide range in
the time taken for the trypsin to react for each individual group. This makes the
data unreliable as the standard deviation shows that there are likely to be
anomalies in the results. Accuracy was not able to be measured properly as the

Maryam Jamshidi

(Core practical)

experiment wasnt repeated, instead, as a class it was done once by each group,
and results were put together to show a range of the results. People tried to be
very accurate, to make a fair class result, but although precision was said to be
carried out, it cannot be proved and therefore not is not very reliable.
The syringe doesnt very accurately measure the volumes of water and trypsin
required and also can gather bubbles within, which will affect the overall volume
of the solution.
The stopwatch may have not been too accurate as time is wasted to start and
stop the clock, which has an impact on the rate of reaction that we write down.