Enzyme activity experiment with liver cells

Name : Maria Camila yañez grade : 10° Biology HL

Lab Guide
Research question :
The aim of this experiment is to
1. Investigate the effect on how much does the conditions that the
enzyme catalase (on liver cells) and the substrate (hydrogen peroxide)
is exposed will have a better enzymatic activity
2. Analyze the effect of the ph. and temperature deviation on the
enzymatic activity
3. Identify the process of efficiency on what level of ph. and temperature
does the enzyme catalase reacts at it’s best with the substrate of
hydrogen peroxide
How does the variation of the ph levels and temperature will affect the efficiency of
the enzymatic activity on the enzyme catalase on length and time?

Hypothesis :
If I exposed the enzyme catalase into six different scenario such as high temperature
(90°c) , low temperature (10°c) a room temperature (19°c) and into a neutral ph of
water (7) , vinegar (ph 2 ) and sodium hydroxide (ph 14) and additionally add 5 ml of
hydrogen peroxide then this conditions will affect the quantity and speed of the
enzymatic reactions because at extreme conditions of temperature and ph will
generate a weakness on the structural hydrogen bonds causing the enzyme to loose
completely the three dimensional shape of the active enzyme causing it do denature
making it impossible for the substrate (hydrogen peroxide) to fit and produce a
chemical reaction. Although on the neutral ph of 7 and the room temperature (19°c)
it is expected to have a the fastest and biggest results because these conditions are
the closest ones to the optimum temperature and ph of the catalase , meaning is the
period were the enzyme works at it’s best.
Background research:
on this experiment it will put putted on the test how the enzyme catalase and the
substrate can work together if it’s exposed to different ph and temperature
conditions. An Enzyme is a globular protein that is in charge of increasing the rate
of a biochemical reaction by lowering the activation energy. The enzyme catalase
reacts to the substrate hydrogen peroxide that decompose to water and oxygen.
The main factors that affect the enzymatic reaction are the ph , temperature and
substrate concentration. At a very high level of temperature the catalase starts to
increment on the frequency of the enzyme-substrate collision and it creates a effect
that it starts to break down the structural hydrogen bonds that from the enzyme
having a product that it changes completely the active site that will no longer fit the
substrate and end up with denaturation. The substrate can be defined a the reactant
of the biochemical reaction , in this case the substrate is the hydrogen peroxide.
Similar to what occurs at extreme temperature it happens on excessive ph levels
were the enzyme is going to pass throughout a process of denaturation. The
optimum temperature of the catalase enzyme is of 36°c because that’s the regular
temperature that animals and humans work with and the optimum ph of the catalase
s 7 do that the liver is exposed to a neutral environment of a ph of these levels.

Materials/equipment:
The materials that are going to be required are:
 pieces of small liver of 2 g each (18 pieces)
 10 drops of hydrogen peroxide (6 times)
 3 ml of Sodium hydroxide (3 times)
 5 ml of vinegar
 5 ml of water
The equipment that is going to be used is
 Test tubes (6)
 Thermometer
 Ruler
 Dropper
 Precision clamps
 5 ml beaker
 Stove
 Gloves
 Lab coat

10 90 Ph Ph Ph
32 7
°c °c 2 14
°c
Methodology
Through this experiment the main concept that is going to be
observed is how efficient are enzymatic reactions of catalase and
hydrogen peroxide on conditions when the temperature and Ph
levels are changed. An enzyme is a globular protein that is in charge
of increasing the rate of a biochemical reaction by lowering the
activation energy.
Test tube temperature 32° c
1. Cut a piece of a beef liver that weight 2 g
2. Use the Precision clamps to pour the liver of room
temperature on the test tube
3. Add 10 droops of hydrogen peroxide
4. Observe and measure how much did the bubbles rise up
5. Repeat this process three times
Test tube temperature 10° c
1. Refrigerate a piece of beef liver that weight 2 g on a temperature of 10°c
2. Use the Precision clamps to pour the liver on the test tube
3. Add 10 droops of hydrogen peroxide
4. Observe and measure with a ruler how much did the bubbles rise up
5. Repeat this process three times
Test tube temperature 90° c
1. Use a stove and pour water
2. Add the piece of beef liver that weight 2 g on the stove till it reaches the
temperature of 90°c
3. Use the Precision clamps to pour the liver of 90°c on the test tube
4. Add 10 droops of hydrogen peroxide
5. Observe and measure with a ruler how much did the bubbles rise up
6. Repeat this process three times
Test tube Ph 7 water
1. Use the Precision clamps to pour the liver on the test tube
2. Add 5 ml of water to the test tube
3. Then add 10 drops of hydrogen peroxide
4. Observe and measure with a ruler how much did the bubbles rise up
5. Repeat this process three times
Test tube Ph 2 vinegar
1. Use the Precision clamps to pour the liver on the test tube
2. Add 5 ml of vinegar to the test tube
3. Then add 10 drops of hydrogen peroxide
4. Observe and measure with a ruler how much did the bubbles rise up and
the time
5. Repeat this process three times
 Test tube Ph 14
1. Use the Precision clamps to pour the liver on the test tube
2. Add 3 ml of sodium hydroxide to the test tube
3. Then add 10 drops of hydrogen peroxide
4. Observe and measure with a ruler how much did the bubbles rise up
5. Repeat this process three times
Variables
Independent: ph levels . and temperature variation
Dependent: efficiency on the enzymatic reaction (time and length)
Control: the amount of hydrogen peroxide , liver samples , test tubes ,
thermometer , ruler
Table : Independent, dependent Variables and control:
DEPENDENT VARIABLE INDEPENDENT

efficiency on the enzymatic reaction Variation on the Ph and temperature

How to manipulate the variable How to manipulate the variable

The efficiency of the enzymatic action The ph and the temperature are going to be manipulated
will be measure by the length of the by a thermometer that will measure the temperature and
amount of bubbles produced on the the Ph variable will be measured on ml with a beaker
enzymatic activity it will be measured
with a ruler in a scale or cm
CONTROLLED VARIABLES

Name of the control variable Equipment How to control de variable

Hydrogen peroxide Dropper This will be measured by the amount of 10
drops will the help of the dropper. I’ll try to
be precise will the number of drops added to
the test tube
Liver examples Electric The live samples will be measured by an
balance electric balance. With this tool I’ll try to
approximate the best that all the samples
weight 2 g
Test tubes NA This test tube will be of the same size,
throughout the whole experiment
Ruler NA This variable will be controlled by using the
same ruler through the whole experiment
Thermometer / chronometer Na This variable will be controlled by using the
same thermometer and chronometer through
the whole experiment
Results
Table of initial raw information
Table data of the temperature variation
Temperature of liver Room temperature Cold liver 10°c Warm liver 90°c
20 °c 18°c 19° c 12°c 9°c 10|c 90°c 90° 90°
Trials #1 #2 #3 #1 #2 #3 #1 #2 #3
Length of the 11 12 9.7 11 6 6 1 0,3 0
enzymatic reaction cm cm cm cm cm cm cm cm cm
(±0.1 cm)
Table data of the ph variation
Ph substances Water ph 7 Vinegar ph 2 Sodium hydroxide
ph 14
Trials #1 #2 #3 #1 #2 #3 #1 #2 #3
Length of the 18 15 20 0 0 0 3 2 2.5
enzymatic reaction cm cm cm cm cm cm cm cm cm
(±0.1 cm)

Table data of the time of enzymatic reaction on temperature

Temperature of liver Room temperature Cold liver 10°c Warm liver 90°c
Trials #1 #2 #3 #1 #2 #3 #1 #2 #3
Time of the 15 s 25 s 20 28 s 27 s 16 s 39 s 29 s 0s
enzymatic reaction s
(±0.1 cm)
Table data of the time of enzymatic reaction on ph
Ph substances Water ph 7 Vinegar ph 2 Sodium hydroxide
ph 14
Trials #1 #2 #3 #1 #2 #3 #1 #2 #3
Time of the 20 s 12 s 35 s 0s 0s 0s 5s 8s 12 s
enzymatic reaction
(±0.1 cm)
Processed Data
Process data of average temperature variation
Temperature Room temperature Cold liver 10°c Warm liver 90°c
19° c
Average of Length 10.9 cm 7.6 cm 0.4 cm
of the enzymatic
reaction (±0.1 cm)
Process data of average Ph variation
Ph Water ph 7 Vinegar ph 2 Sodium
hydroxide ph 14
Average of time of 17.6 cm 0 cm 2.5 cm
the enzymatic
reaction (±0.1 s)
Process data of average time reaction on temperature
Temperature Room temperature Cold liver 10°c Warm liver 90°c
19° c
Average of time of 22.6 s 23.6 s 24.6 s
the enzymatic
reaction (±0.1 s)
Process data of average time reaction on Ph
Ph Water ph 7 Vinegar ph 2 Sodium
hydroxide ph 14
Average of time of 22.3 s 0s 8.3 s
the enzymatic
reaction (±0.1 cm)

Graph of length vs time of enzymatic reaction on the variable of temperature

length vs time of enzymatic reaction Standard deviation :

(temperature)
Time : 1

25 Length : 5,25
20
15
10
5
0
1 2 3
lenght 10.9 7.6 0.4
time 22.6 23.6 24.6

lenght time
 Graph of length vs time of enzymatic reaction on the variable of temperature

lenght vs time of enzymatic reaction (Ph) Standard deviation :

Time : 1,8
25
20 Length : 2,5
15
10
5
0
1 2 3
lenght 17.6 0 2.5
time 22.3 0 8.3

lenght time

Observations

on this image it can be observed the enzymatic reaction of the

liver cells that are a room area temperature of 19°c. as it’s shown
on the picture there was a instant and big reaction of the enzyme
catalase with the substrate hydrogen peroxide. This is because
the catalase brake down the hydrogen peroxide to oxygen (which
are the bubbles) and water. the temperature was appropriate for
the reaction to occur since it a big reaction a small time.

On the following picture it can be observed the liver cells that

were on a temperature of 10°c. As seen it can be stated that that
since the enzyme was exposed to a lower temperature it slowed
down the process of the enzymatic reaction causing harder for the
enzyme to break the hydrogen bonds, although it didn’t reach a
limit were the enzyme denatured.
 this picture shows the liver at a temperature of 90°c .
through the three different trails the enzymatic
reactions were barely noticeable. This can be caused
by the result o denaturation on the catalase enzyme.
Do that the temperature was very high the enzyme lost
completely it’s shape and couldn’t react with the
substrate.

Here is the liver that was exposed to a level of ph 7

(water) . the result for this were positively high since
this level of ph that was exposed is the optimal ph of
this enzyme to react. the enzymatic reactions were
fast, and it was generated in a big quantity.

On this image it can be observed how the catalase

reacts to being exposed on a acidic solution. Since the
optimal ph is 7 there were no reactions do that the
vinegar had a ph of 2 making the enzyme to complete
denature immediately and not caused any enzymatic
reaction.

The scenario that the catalase was exposed was to the

base of sodium hydroxide that has a ph of 14. This factor
created that the enzymatic reaction was very low and
slowed the process. This little reaction is because the
enzyme is not able to break down the hydrogen bonds.
Data processing
Throughout this experiment the data that was recollected was the length of the
enzymatic reaction and time it took the enzyme to generate the biochemical process.
These two variables were taken into a measuring process because with the amount
of bubbles the chemical reaction were made it it’s proven how much efficient is the
enzymatic reaction. Also, the time because it shows fast is the enzyme able to do
this process. The raw data tables were taken in two ways the first one was measuring
the length of how much did the bubbles rise up on the test ( the instrument that was
used was a ruler) , and the time was taken through a chronometer form the time the
hydrogen peroxide was added to the test tube till the reaction stopped. Then the
process data was obtained by adding the three trails of the different temperature and
ph levels and divided by three, with this it was able to find the average length and
time it took the enzyme catalase to generate it’s enzymatic activity. With this
information there were two graphs made with the objective to compare the length vs
the time of the reaction that are affected do to the temperature and ph variation.
Analysis
The enzyme activity experiment was based on factors two main factors that are the
time that lasted the enzyme activity and the quantity that it had. these variables were
chosen because this help to identify the enzymatic action. This test was based on
six different scenarios were the enzyme catalase (find in liver cells) was going to be
exposed on a room temperature of 19°c , then added a quantity of 5 ml of hydrogen
peroxide. As seen in the tables and the images this had a fast and big enzymatic
reaction, it can be proven by the processed data of temperature average that it has
the fastest time to produces this reaction and in a big quantity of 10.9 cm and a time
of .20s . This were results this way because the optimum l temperature of the enzyme
catalase is of 36° c , on comparison to the other temperature of 10° and 90° this was
the one closest to the optimal temperature. Since the enzyme was exposed to a
good temperature , the hydrogen peroxide entered to the active site of the catalase
and was able to interact with the chain of the amino acids , causing a proton to
transfer between the oxygen atoms. These free oxygen atoms coordinate an action
to create a water molecule that reacts with another hydrogen peroxide molecule that
produces water and oxygen, on this case the oxygen are the bubbles that evidence
that this biochemical process occurred.
On the second scenario were the catalase enzyme was exposed to a low
temperature of 10° c surprisingly the enzymatic reaction was presented in a high
rate considering the conditions the enzyme had been exposed with a length of 7.6
cm and a time rate of 23.6 s. for this enzyme the optimum temperature is of 36° c
and this enzyme was on 10° it was expected that the chemical reaction would have
been minimal do that at low temperature molecules move more slowly , generating
a reduction on the frequency of enzyme-substrate collisions , therefor it decreases
completely the enzyme activity. Although in this case it may have made the process
more slower and the production was less, but it didn’t get to a limit that desaturated
the enzyme. On the table of processed data this was the second fastest and biggest
enzymatic reaction, this results could occurred because this particular enzymes on
the liver had the ability to keep the enzyme- substrate collision.
On the third scenario the liver was exposed to a high
temperature of 90°c, as seen on the process data of the
variation of temperature there was a length of 0,4 cm with a
time rate of 22.3 s. this result were this way because the
catalase was on a very high temperature of 90° (close to a
boiling point) creating that the chemical structures and
bonds that held together ,starts to break down. The process
of starting to loss the original structure it causes the enzyme
to no longer have the original shape it had , make the active
site different. Denaturation no longer admits that the
substrate fits properly, just like on the experiment, it is
inferred that the mayor enzymes were desaturated do to the
high temperatures that was exposed , generated a lack of
enzymatic activity.
On the fourth scenario that involved the ph factor, the catalase enzyme was
exposed to 5 ml of water and 5 ml of hydrogen peroxide. Observing the processed
data of average variation of ph levels, the water was the highest and fastest form the
other two. This is because the optimum ph level of the catalase is of 7 , meaning is
the period were the enzyme works at it’s best. When this occurred the enzyme had
the ability to break down the hydrogen bonds causing a fast biochemical reaction.
As it’s seen on the process data of ph variation it had a length of 17.6 cm and a time
rate of 22.3 s , in comparison with the other two ph levels this was the most efficient
one because of the ph condition that was optimum for the enzyme to react.
On the fifth scenario the liver was exposed to an acidic solution meaning that on the
test tube it was added 5 ml of vinegar that had a ph of 2 as it can be observed on
the ph strand strips that are labeled with the color red to represent this level of ph.
during the experiment there was no reaction at all , this can be evidence by the
processed data table of average ph variation. On this scenario there was 0 cm length
and 0s of time rate. This results were given this way because the enzyme completely
denatured , when this process occurs the enzyme completely looses it’s original
three dimensional because of the break down of the structural hydrogen bonds that
generate a change of shape on the active site , making it impossible for the substrate
(in this case hydrogen peroxide) to create a enzymatic reaction.
On the last scenario the enzyme catalase was exposed to a bas solution, meaning
that on the test tube there was added 3 ml of sodium hydroxide , with 5 ml of
hydrogen peroxide on the test tube. Observing the process data table there was
barely a reaction present. The base that was used was Sodium hydroxide that
contains a ph of 14, this is verified by the ph strand strip that are represented with a
dark blue that represent the ph of 14. On the process data table of average ph
variation this scenario had a length of 2.5 cm and a time rate of 8.3 s. it is inferred
that the majority of the enzymes denatured because of the environmental conditions
and chemical conditions, since the optimum ph level is of 7 (water) it was exposed
to a level in which it was two times higher. The extreme base solution generated the
enzyme lost completely it’s structure causing the structural hydrogen bonds to
weaken a lessen completely to a point were the active site no longer could fit with
the substrate.
Conclusion
The enzyme reaction experiment was based on the analysis and
investigation on how much does the temperature on distinct stages
and the variation of ph levels affect the speed and quantity of the
enzymatic reaction on the enzyme catalase (found in liver cells) with
the substrate of hydrogen peroxide. The main aim of this experiment
was to be able to observe and identify how much effect did the factors
of temperature and ph had on the enzyme. The main purpose of using
the enzyme catalase during this experiment was because in
comparison with other enzyme there was a way to measure on a more
amplified way the enzymatic reaction through length and time. The question o this
investigation was “ How does the variation of the ph levels and temperature will affect
the efficiency of the enzymatic activity on the enzyme catalase?” to which the
experiment had the results that on the temperature that was exposed at 10° the
enzymatic reaction was surprisingly high contradicting the hypothesis that stated
that at a extreme temperature the enzyme will denatured and not produce any
enzymatic reaction. This can be proven by the data on the process data of variation
of temperature that shows it had an average length of 7.6 cm and a time rate of 22.6.
it can be inferred that the temperature may affect the process of the enzymatic
reaction and slowed the process because the temperature slows down the frequency
of enzyme-substrate collisions making a lesser production.
At a room temperature of 19° the enzyme the biochemical reaction was the highest
and fastest in comparison of the other two temperatures of 90°c and 10°c. the results
were of 10.9 cm on a period of 22.6 s this is because the optimum temperature of
the enzyme catalase is of 36°c, since the room temperature of 19° was the closest
to this point it had a good environmental conditions in which the hydrogen peroxide
was able to entre to the active site and interact with the amino acids , causing a
proton to transfer into oxygen atoms. This free oxygen atoms coordinate a process
in which as a result they form a water molecule and oxygen. With this evidence it
can be stated that the hypothesis is accepted because as it was stablished before
the room temperature was the one to achieve a better enzymatic action.
At a elevated temperature of 90°c the enzyme catalase reaction was very low it had
a length of 0.4 cm and a rate of time of 24.6 s, as expected on the hypothesis it can
be declared that it is accepted because the high temperature affected the enzyme
activity to decrease in comparison to the other temperatures. It is inferred that the
majority of the enzymes went through a process of denaturation, in which the heat
breaks down the hydrogen bonds that make up the enzyme structure affecting the
active site and changing it’s shape , making it impossible for the substrate to fit in.
do to this factors the enzyme couldn’t make a efficient biochemical reaction.
When the enzyme was exposed to a acidic solution of ph of 2 (vinegar) the reaction
was nule , throughout the three trails there was no evidence of enzymatic activity
making the hypothesis accepted because it was said that at a extreme levels of ph
the catalase had a bigger probability to be affected by denaturation. It is stated that
all the enzymes inside the liver at a ph of 2 were affected on their three dimensional
shape creating a change on the active site , making it impossible for the substrate to
fit in and generate a chemical reaction. This reaction was similar to what occurred
on the enzyme that was exposed to a ph of 14 on sodium hydroxide which had
results of a length 2.5 cm and a time rate of 8.3 s. just as the previous scenario the
enzyme was exposed to a acid environment the same process occurred with the
basic solution that it may have desaturated the majority of the enzyme affecting the
hydrogen structural bonds that made a impact on the active site of the enzyme.
Although in comparison the other the enzyme that was exposed to a ph of 2 , it was
able to still produce an enzymatic reaction. With this evidence it can be stated that
the hypothesis stated previously is not completely accepted do that it didn’t had a
mayor efficiency but it still throughout all of the ph factor it was able to generate the
biochemical process.
On the last scenario the catalase was exposed to a neutral ph of 7 (water) as it is
observed on the table of processed data of average ph it can be evidence that in
comparison to the acid and base it had the fastest enzymatic reaction and in a big
quantity of , just as the hypothesis that was declared previously that since the
optimum ph of catalase is 7 it was able to have a effective enzymatic reaction were
the hydrogen peroxide enter to the active site and as a product oxygen and water is
generated. The optimum ph is 7 because of the environmental conditions that the
liver is surrounded it’s ph environment regularly is of 7.
Evaluation
During the experiment the variable of length could have been taken into another way
of recollecting the quantity of the enzyme activity, because even though the length
was calculated with the measurements of the ruler there were few times when the
enzymatic reaction was too high that the bubbles spread out side of the test tube, so
for the next experiment to have a more precise data I’ll look for another strategy of
measurement. Another factor that could have affected was the time since the liver
was in a constant change of temperature it may have affected the enzyme activity.
This can be improve by having a bigger lapse of time.
Bibliography

Buddies, Science. “The Liver: Helping Enzymes Help You!” Scientific American, 8 Mar. 2012,
www.scientificamerican.com/article/bring-science-home-liver-helping-enzymes/.

Smith, Brett. “How Does Temperature Affect Catalase Enzyme Activity?” Sciencing, 14 Apr. 2017,
sciencing.com/temperature-affect-catalase-enzyme-activity-7776025.html.

Smith , M. (2014, August 4). GCSE Bitesize: Temperature, pH and enzymes. Retrieved January 22,
2018, from
http://www.bbc.co.uk/schools/gcsebitesize/science/add_aqa/proteins/proteinsrev3.sh
tml

Graw, M. (2017 , April 25). What Are the Effects of Boiling & Freezing on Enzyme Activity?
Retrieved January 22, 2018, from https://sciencing.com/effects-boiling-freezing-enzyme-
activity-23207.html