Zach Anderson
Edda Cammick
Biology 113
8 October 2014
Enzyme Functionality Under Different Conditions Such as in the
Presence of an Inhibitor, Exposure to Extreme Temperature, and
Environments With Varying pH Values
Abstract:
A series of experiments were conducted to observe how the enzymes
amylase and catechol oxidase work. The hypothesis that catechol oxidase would
catalyze the reaction between catechol and a potato slice was supported. It could
be observed because this reaction caused the potato slice to turn a darker
shade. It was also observed that since catechol oxidase needs copper as a
cofactor, the use of an inhibitor that binds to copper would cause the enzyme to
be unable to function. The enzyme amylase was found to be unable to function if
exposed to extreme temperatures and or an acidic or basic pH environment.
Introduction:
Enzymes are biological catalysts that speed up chemical reactions and
are inexhaustible. They can increase the rate at which reactions occur by up to a
factor of 10^19. The material that is being acted on by the enzyme is called a
substrate. Enzymes work because they can bind to the transition state better
than the substrate, which lowers the activation energy causing the speed of a
reaction to accelerate. The enzyme combines with a substrate at the enzymes
active site which creates the enzyme substrate complex. This is where catalysis
takes place. When the reaction is complete, the complex dissociates. After the
complex dissociates the enzyme is unchanged and as a result can move on to
catalyze another reaction. Even though enzymes speed up reactions they do not
have the ability to determine the direction of the reaction.
Enzyme activity can be affected by many external factors including
temperature and pH. For the enzyme amylase, which is involved in the
breakdown of starch into glucose and is found in the mouths of humans and a
few other mammals, the temperature cannot be too hot or too cold or else it will
become denatured and cannot function. The same is true of pH. Amylase cannot
function if exposed to a pH that is too acidic or basic.
The enzyme catechol oxidase in the presence of oxygen catalyzes the
removal of electrons and hydrogens from catechol causing it to be converted to
benzoquinone, which polymerizes to form catechol melanin. This melanin causes
the darkening of fruits and vegetables therefore when catechol oxidase comes
into contact with a fruit or vegetable it turns a darker color.
To become active catechol oxidase requires copper as a cofactor. This
means that it can be turned ineffective if exposed to an inhibitor. The inhibitor
phenylthiourea (PTU) commonly combines with copper. If this occurs the enzyme
catechol oxidase is rendered useless.
Hypothesis:
For the following the first sentence is the experimental hypothesis and the
second is the null.
4.1) If catechol oxidase is exposed to both catechol and the potato slice then a
reaction will occur that turns the potato slice a darker color. If both the potato
slice and catechol are not present then the reaction will not occur.
4.2) If catechol oxidase is exposed to the inhibitor PTU in the same concentration
then the reaction will not occur. If there is less PTU then catechol oxidase then a
reaction will occur.
4.3A) The higher the concentration of amylase the faster a reaction will occur.
The lower the concentration of amylase the faster a reaction will occur.
4.3B) If amylase is exposed to an environment where the pH is acidic or basic
then a reaction will not occur. If amylase is exposed to an environment where the
pH is acidic or basic then a reaction will occur.
4.3C) If amylase is exposed to extreme cold or hot temperatures then it will
denature and a reaction will not occur. If amylase is exposed to extreme cold or
hot temperatures then it will not denature and a reaction will occur.
Materials and Methods:
To begin the first experiment three experimental tubes are prepared
containing the same total amount of solution. The pipettes cannot be
contaminated. After each tube is prepared, a finger was used to hold a Parafilm
square securely over the tube mouth. Then the tube was rotated to mix the
contents thoroughly. A fresh square was used for each tube. The contents of the
tubes are as follows:
Tube
Distilled Water Catechol
Distilled Water Potato Extract
1
5mL
0.5mL
0.5mL
----
2
5mL
0.5mL
----
0.5mL
3
5mL
----
0.5mL
0.5mL
The results were recorded after the reactions in the tubes were observed.
For the next experiment another three tubes were prepared using the
guidelines in the following table:
Tube
Distilled
Potato
PTU
Distilled
Catechol
Water
Extract
1
5mL
0.5mL
0.5mL
0.5mL
0.5mL
2
5mL
0.5mL
0.5mL
----
1mL
3
5mL
0.5mL
----
1mL
0.5mL
Water
It is important to note that catechol was added to the tubes last. The results were
recorded after the reactions in the tubes were observed.
For the next experiment an amylase dilution was prepared. 5 standard test
tubes were numbered 1-5. Using a 5mL-graduated pipette, 5mL of distilled water
were added to each tube. Then several dilutions were made with different
concentrations of amylase. Then experimental test tubes were prepared. A
second set of 5 test tubes was then numbered 1-5. Beginning with tube 5 of the
first set, 2mL of the dilution was transferred into tube 5 of the second set. A 5 mL
pipette was used for the transfer. The pipe was then rinsed in distilled water and
the procedure was repeated for tubes 4,3,2, and 1. After these transfers test tube
set 1 was no longer needed. Then 2mL of pH 6.8 buffer solution was added to
each of the tubes in the second set. Then 1-2 drops of I2KI were added to each
compartment of four rows of a test plate. The rate at which the each reaction
occurred was then recorded.
For the next experiment 6 standard test tubes were numbered 1-6 and
marked with a pH value of 4, 5, 6,7,8, or 9. Then 5mL of the appropriate buffer
was added to each of the tubes. The pipette was rinsed with distilled water after
dispensing each buffer. Then 1.5mL of amylase was added to each tube and
mixed in. 1 or 2 drops of I2KI were then placed in the compartments of several
rows of the test plate. Then using only tube 1, 2.5mL of the 1% starch solution
was added. This time was then recorded as 0. A drop of the reaction mixture
from tube 1 was then removed and added to a drop of I2KI on the test plate. The
reaction mixture was then sampled at 10-second intervals until a blue color was
no longer produced. The time it took for the digestion of the starch was then
recorded. Then the same process was repeated for the remaining 5 test tubes.
For the next experiment, 4 standard test tubes were numbered 1-4. Then
using a 5mL-calibrated pipette, 2mL of the 1% starch solution was added to each
tube. Next 4 mL of DI water was added to each tube. Then 1 mL of 6.8 buffer
was added to each tube. The 4 tubes were then placed as follows:
1) 80 degrees Celsius water bath
2) 37 degrees Celsius water bath
3) Test tube rack
4) Beaker of crushed ice
A second set of 4 test tubes were then marked 1A through 4A. Then 1 mL of
amylase was added to each tube and placed in the same manner as the first set
of tubes respectively. All 8 tubes were then allowed to sit for 10 minutes. Several
rows of the test plate were then filled with 1 or 2 drops of I2KI per compartment.
Tubes 1 and 1A were then mixed. The mixture was then added to a drop of I2KI
on the test plate. The mixture was then added to a new well at 30-second
intervals until a blue color was no longer produced and the I2KI solution
remained a yellow-amber color. The time it took since the first drop was placed
was recorded. The same steps were repeated for each of the other 3 pairs of test
tubes.
Results:
Table 4.1
Test Tube
Start Color
Finish Color
1
Light yellow/ clear
Light yellow/ clear
2
Light yellow/ clear
Dark yellow/ clear
3
Clear
Clear
Test Tube
Start Color
Finish Color
1
Yellow/ clear
Peachy/ clear
2
Yellow/ clear
Light green/ clear
3
Yellow/ clear
Olive Oil yellow
Percent of Amylase (%)
Time of Starch
Table 4.2
Table 4.3A
Disappearance
1
0.50
0 sec
2
0.25
0 sec
3
0.125
0 sec
4
0.063
40 sec
5
0.031
70 sec
% of amylase
0.6
0.5
0.4
0.3
% of amylase
0.2
0.1
0
0
0
0
40
70
Table 4.3B
Test Tube
pH
Time of Starch
Disappearance
1
4
Termination
2
5
Termination
3
6
6:20 min
4
7
5:00 min
5
8
Termination
6
9
Termination
Table 4.3C
Test Tube
Degrees C
Time of Starch
Disappearance
1
80
No reaction
2
37
0 min
3
22
0 min
4
4
No reaction
Conclusion:
In experiment 4.1, the effects of catechol oxidase were observed. The
results of the experiment can be found in table 4.1 above. Test tube 2 was the
only tube that changed color as a result of the reaction brought on by catechol
oxidase. This reaction occurred because the catechol in this tube only because it
was the only tube that contained both the potato extract and the catechol. The
reason the other tubes did not see a change was because the potato extract and
the catechol were not present at the same time.
In experiment 4.2, the effects of inhibitors on the enzyme catechol oxidase
were observed. The specific inhibitor used was called phenylthiourea (PTU). The
PTU inhibits catechol oxidase by combining with copper that is a cofactor for the
enzyme. In the first test tube the amount of catechol was the same as the
amount of PTU, which resulted in the reaction not taking place at all. Since there
was twice as much catechol as PTU in the second tube the reaction was still able
to take place only it was slower. In the third tube there was no PTU present,
which allowed for the reaction to be completed entirely.
In experiment 4.3A the effects of different concentrations of amylase on a
reaction were observed. The results presented in table 4.3A show that the higher
the concentration of amylase the quicker the reaction took place.
In experiment 4.3B the effects of pH on Amylase function were observed.
The tubes with the pH’s of 4, 5, 8, and 9 did not show a sign of amylase activity.
The tubes with the pH’s of 6 and 7 however showed signs that the amylase was
acting on the starch. The tube with pH of 7 is the most neutral and therefore is
the optimal pH for amylase to act.
In experiment 4.3C the effects of temperature on the function of amylase
were observed. When exposed to extreme cold and hot temperatures the
amylase denatured and did not react. When exposed to room temperature and at
37 degrees Celsius, the amylase did not denature and was therefore able to
react with the starch.
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