Lab Substrate Concentration & Enzymes
Introduction:
Enzymes are biological catalysts. They are generally large proteins made up of several
hundred amino acids. Enzymes catalyze thousands of chemical reactions that occur in living
cells. Enzymes are highly specific so each one speeds up only one particular chemical
reaction. Many kinds of enzymes are found in each cell but because they are used over and
over there may be only a small amount of each enzyme present.
In this lab activity, you will study the enzyme catalase and its substrate hydrogen peroxide.
Catalase accelerates the breakdown of hydrogen peroxide (H2O2) into water (H2O) and oxygen
(O2). The chemical equation for this reaction is:
2 H2O2 ----Catalase-------> 2H2O + O2
Catalase is found in both plant and animal tissues. It is especially abundant in plant storage
organs such as potatoes and the fleshy parts of fruits. Catalase is extremely important in cells
because it prevents the accumulation of hydrogen peroxide. Hydrogen peroxide is a strong
oxidizing agent, which tends to disrupt the delicate balance of cell chemistry. If too much
hydrogen peroxide accumulates, it will kill the cell.
Several factors affect the action of enzymes: salt concentration, pH, temperature, enzyme
poisons, radiation, the concentration of enzymes, and the concentration of the substrate.
This lab deals only with how an enzyme is affected by different concentrations of substrate specifically, what effect will different concentrations of a substrate (H2O2) have on an enzyme
(catalase in Pig Liver).
OBJECTIVES
In this experiment, you will
 Use an Oxygen Gas Sensor to measure the production of oxygen gas as hydrogen
peroxide is destroyed by the enzyme catalase or peroxidase on substrate concentrations.
 Measure and compare the initial rates of reaction for this catalases enzyme when different
concentrations of Substrate are used.
Safety Notes:
1. Hydrogen peroxide can damage your clothes. Rinse any spills with water immediately.
2. Keep hydrogen peroxide out of your eyes. Wear safety glasses!
3. Report any accidents of spills to your instructor.
1
NAME: Josephine Kooij
Date: March 12, 2014
Title: Investigating how substrate concentration affects enzymatic activity through the
examination of oxygen (ppm) production as a result of catalase’s decomposition of hydrogen
peroxide to oxygen and water
Research question:
How do different concentrations of hydrogen peroxide (2H2O2) affect catalase’s rate of
enzymatic activity through measuring the oxygen produced (ppm)?
Hypothesis:
Experimental hypothesis:
Due to extensive background research, it seems that as the concentration of hydrogen peroxide
increases, the enzymatic activity of catalase will increase as well, until a plateau occurs due to
the saturation of the enzyme. As the liver cube used for experimentation is 1 cm3 and the
highest hydrogen peroxide concentration is 3.0%, it seems we will not see a plateau phase just
yet, and instead will observe a positive trend. As the substrate concentration increases, so will
the enzymatic activity (oxygen production/time being a reflection of this—more oxygen
produced over time, the higher the rate of reaction and vice versa).
Null hypothesis:
Varying substrate concentrations will have no affect on enzymatic activity of catalase in
breaking down hydrogen peroxide into water and oxygen gas.
Variable Table:
Independent variable
Dependent variable
Hydrogen peroxide concentration
(Percentage of Hydrogen peroxide in 10±0.2
ml solution)
Rate of enzymatic action through measuring
the amount of oxygen released as product of
reaction (ppm (parts per million with
uncertainty of ±1%)
Control Variables
1
10±0.2 ml of each different hydrogen
peroxide concentration
2
Temperature
3
From which animal the liver is obtained and
the size of the liver
4
pH level
5
Time
Control Group
0.0±0.1% hydrogen peroxide concentration
Materials: (per group)
25 ml graduated cylinder
2
30ml beaker
Hydrogen peroxide (0%, 0.1%, 0.5%, 1%, 3%)
Forceps
Ruler
Cube of Liver (1cmX1cmX 1cm)
Scalpel
Cutting Board
Vernier O2 Gas Sensor
400 mL beaker
10 mL graduated cylinder
Three 18  150 mm test tubes
250 mL Nalgene bottle
Procedure:
1. Obtain and wear goggles.
2. Connect the O2 Gas Sensor to Lab Quest and choose New from the File menu. If you
have an older sensor that does not auto-ID, manually set up the sensor.
3. On the Meter screen, tap Rate. Change the data-collection rate to 0.2 samples/second
and the data-collection length to 180 seconds.
4. On cutting board cut a cube of liver (1cmX1cmX 1cm)
5. Obtain H2O2 concentrations (0%, 0.1%, 0.5%, 1%, 3%)
6. Pour 10ml of the 0% hydrogen peroxide into the 250 mL Nalgene bottle. Place the O2
Gas Sensor into the bottle as shown in Figure 1. Gently push the sensor down into the
bottle until it stops. The sensor is designed to seal the bottle with minimal force.
7. Drop Liver cube into
8. Start data collection (O2 Production in parts per million (ppm) for 180 seconds.
9. When data collection is complete, a graph of O2 gas vs. time will be displayed. Remove
the O2 Gas Sensor from the Nalgene bottle. Rinse the bottle with water and dry with a
paper towel.
10. Perform a linear regression to calculate the rate of reaction.
a. Choose Curve Fit from the Analyze menu.
b. Select Linear for the Fit Equation. The linear-regression statistics for these two data
columns are displayed for the equation in the form
y = mx + b
c. Enter the absolute value of the slope, m, as the reaction rate in Table 1.
d. Select OK.
9. Repeat steps 1-8, four more times using the other hydrogen peroxide solutions (0.1%,
0.5%, 1%, 3%) and a new Liver cube for each trial
10. Store the data from the first run by tapping the File Cabinet icon
11. Tap Run four, and select All Runs. All three runs will now be displayed on the same
graph axes.
12. Obtain class data for each concentration of hydrogen peroxide used by all groups in the
class and record this data in Table II.
3
Process Data:
Raw Data (DCP Aspect 1)
Table 1: Rate (%/s) of oxygen produced over time for different concentrations of H2O2 as a
reflection of the rate of catalases activity when decomposing H2O2
Hydrogen peroxide (H2O2) concentration
0.0±0.1%
H2O2
0.1±0.1%
H2O2
0.5±0.1%
H2O2
1.0±0.1%
H2O2
3.0±0.1%
H2O2
Group 1**
-0.001180
-2.812E-05
0.002677
0.003413
0.03579
Group 2
-0.002800
-7.850E-5
0.0004275
0.003581
0.01295
Group 3
-7.505E-5
-0.000139
0.001077
0.0009798
0.009824
Group 4
-0.0008587
0.000445
0.001613
0.004353
0.0266
Group 5
-0.001534
-1.733E-5
0.001533
0.003307
0.02685
Group 6
-0.001007
-0.0007415
0.0005470
0.002857
0.02550
Group 7
-0.0007647
-9.300E-05
0.004019
0.005190
0.01095
Group 8
-0.001218
-0.0004672
0.0003569
0.007518
0.009326
Group 9
-0.0000112
0.0007780
0.002078
0.008162
0.02220
Group 10
-0.001057
0.0002641
0.0001087
0.007616
0.03206
**Our group data
Observations:
Justification of uncertainty
The uncertainty for the hydrogen peroxide concentration, ±0.1%, was given to us by the lab
technician.
The uncertainty for measuring oxygen parts per million, ±1%, was give to us by the lab
technician.
The uncertainty for the measurement of the graduated cylinder is ±0.2 ml, as it was a 10 ml
graduated cylinder with the smallest increment of 0.2 ml.
The uncertainty for the ruler used to measure 1 cm 3 is ±0.05cm. Ruler: (0.1cm)/2 = ± 0.05cm
Outliers
No outliers were included as each of the values for the different concentrations of hydrogen
peroxide followed a general pattern and stayed within 2 standard deviations of the mean. The
only concentration that led to major variations in the data seems to be the 3% hydrogen
peroxide concentration. It is however not possible to remove data points as there is not a
majority amount of data points in one area. The data points are spread fairly evenly above and
below the average value and are also within 2 standard deviations of the mean. Therefore I
have not identified any outliers.
4
Observations
It became clear that as the concentration of hydrogen peroxide increased, the amount of white
bubbles covering the surface of liver would increase. The cube engorged in 0% hydrogen
peroxide showed barely any bubbles on the surface. The cube was also slightly larger than the
others. The cube engorged in 0.1% hydrogen peroxide was slightly smaller and had a little more
bubbles. This pattern became more evident as the concentration of hydrogen peroxide
increased from 0.1% to 0.5%, 1%, and finally 3%. The cube engorged in 3% hydrogen peroxide
was covered in a significant amount of bubbles and was clearly releasing the most amount of
oxygen. As the bubbles contain oxygen being expelled as a product of catalases enzymatic
activity, we were able to use these observations as corroboration for our data collection.
Processed Data (DCP Aspect 2)
Table: Rate (%/s) of oxygen production as catalase decomposes different concentrations of
H2O2 overtime
Hydrogen peroxide (H2O2) concentration
0.0±0.1%
H2O2
Group
1**
0.1±0.1%
H2O2
0.5±0.1%
H2O2
1.0±0.1%
H2O2
-0.00118
-2.81E-05
0.002677
0.003413
Group 2
-0.0028
-7.85E-05
0.0004275
0.003581
Group 3
-7.51E-05
-0.000139
0.001077
0.0009798
Group 4
-0.0008587
0.000445
0.001613
0.004353
Group 5
-0.001534
0.001533
0.003307
Group 6
-0.001007 -0.0007415
0.000547
0.002857
-9.30E-05
0.004019
0.00519
-0.001218 -0.0004672
0.0003569
0.007518
Group 7
Group 8
Group 9
Group
10
Average
-0.0007647
-1.73E-05
-0.0000112
0.000778
0.002078
0.008162
-0.001057
0.0002641
0.0001087
0.007616
0.001050565
-7.76E-06 0.00144371 0.00469768
R value
(Correlation
test)
0.97675589
0.03579
4
0.99076327
0.01295
3
0.97552368
0.009824
3
0.98682107
0.0266
6
0.98373047
0.02685
9
0.98157433
0.0255
2
0.96162541
0.01095
9
0.88226229
0.009326
3
0.99564561
0.0222
6
0.98986545
0.03206
9
0.99550802
0.021205
3
3.0±0.1%
H2O2
Standar
d
0.00043150 0.00122595 0.00237757 0.00974628
deviation 0.000781125
6
7
1
2
5
Presenting Processed Data (DCP Aspect 3)
Graph 1: Rate (%/s) of oxygen production as catalase decomposes different concentrations of
H2O2 overtime
As this graph is a cummulation of all data collected within the experiment, it clearly shows how
each of the groups data compares to each other and how the different solute concentrations
affected the rate of oxygen production. Each of the groups seem to be fairly close in values and
there doesn’t seem to be much variation, except for the values collected for the concentration of
3% hydrogen peroxide. It is clear that the values collected for this hydrogen peroxide
concentration are, although clearly higher in value than the other concentrations of hydrogen
peroxide, spread out throughout around 0.01%/s to 0.04%/s (oxygen production). This is
discussed later as standard deviations and averages are collected in Graph 3.
6
Graph 2: Average rate of oxygen production (%/s) for different concentrations of H 2O2 as
catalase decomposes H2O2 overtime
This graph clearly shows the correlation between the two variable, the hydrogen peroxide
concentration and the rate of oxygen production. As the R value is 0.995508023 and the R2
value is 0.99104, it seems clear that a very strong positive correlation is observed. This upward
trend means that as the substrate (hydrogen peroxide) concentration increases, the rate of
oxygen production, as a reflection of the enzymatic activity, increases as well.
7
Graph 3: Average rate of oxygen production (%/s) for different concentrations of H2O2 as
catalase decomposes H2O2 overtime
This graph clearly shows the range within the data sets. As it shows the average oxygen
production for each hydrogen peroxide concentration, it gives an accurate view of the general
standard deviation (represented by error bars) for each group. The error bar that immediately
stands out as the largest is that of the 3.0% hydrogen peroxide concentration. This proves that
a large amount of variation occurs between the data points collected by different group. This
makes the data shown on the graph less reliable. The error bar of the 1.0% hydrogen peroxide
concentration is the second largest and the size of the error bars continues to decrease up until
0.1% hydrogen peroxide. As less substrate concentration is added to the solution, less variation
8
seems to occur in the data, until the 0.0% hydrogen peroxide concentration, which is only
slightly bigger than the 0.1% hydorgen peroxide concentration. None of the error bars are
however overlapping with the 3.0% hydrogen peroxide error bar, meaning there is a significant
difference between it and the other hydrogen peroxide concentrations. There is not much
overlapping of the other groups aswell and the t-tests below showed there was still a sgnificant
difference between 0.5% (overlapping slightly with the 1.0% and 0.10% hydrogen peroxide
concentrations) and all other concentrations, proving all data sets are significanty different.
T Test
A T test was performed on data sets that were thought to emphasize extremes and in this way
increase the ability to draw significant conclusions.
Comparison of rate of oxygen production (%/s) as catalase decomposes 0.0% hydrogen
peroxide and other concentrations
As the solution with 0.0% hydrogen peroxide is the control group it would be useful to compare
this group with the other groups with different hydrogen peroxide concentrations.
Null hypothesis: there is no significant difference in the means of the data sets.
Comparison
hydrogen
peroxide
concentrations
T-value
P-value
Degree of
Freedom
Critical Value Reject or
Accept Null
Hypothesis?
0.0% and 0.1%
3.6953
0.0017
18
2.10
Reject as the
t-value is
greater than
the critical
value and the
p-values is
less than 5%.
0.0% and 0.5%
5.4260
Less than
0.0001
18
2.10
Reject as the
t-value is
greater than
the critical
value and the
p-values is
less than 5%.
0.0% and 1%
7.2635
Less than
0.0001
18
2.10
Reject as the
t-value is
greater than
the critical
value and the
p-values is
less than 5%.
9
0.0% and 3%
7.1980
Less than
0.0001
18
2.10
Reject as the
t-value is
greater than
the critical
value and the
p-values is
less than 5%.
Comparison of rate of oxygen production for decomposition of 0.1% H2O2 and 0.5% H2O2
It seemed to me interesting to compare values that were situated in the middle of the range of
independent variables, as this might show just how little of an increase in substrate
concentration it might take to make a significant difference.
Null hypothesis: there is no significant difference in the means of the data sets.
Degree of freedom: 18
Critical value: 2.10
P value: 0.0024
T value: 3.5316
Conclusion: Reject the null hypothesis as the t-value is greater than the critical value and the pvalues is less than 5%.
Comparison of rate of oxygen production for decomposition of 0.5% H2O2 and 1% H2O2
I chose to compare the rate of oxygen production for these two concentrations as they are also
in the middle of the data and wished to prove the in comparison these groups might also have a
significant difference.
Null hypothesis: there is no significant difference in the means of the data sets.
Degree of freedom: 18
Critical value: 2.10
P value: 0.0012
T value: 3.8467
Conclusion: Reject the null hypothesis as the t-value is greater than the critical value and the pvalues is less than 5%.
10
Sample Calculations for Processed Data
Slope/rate of
change
The Rate (%/s) of oxygen produced over time for different concentrations
of H2O2 was measured using an oxygen probe attached to Logger Pro.
After collecting data using Logger Pro, one would then go to Analyze and
Curve fit line, where they would be able to deduce the slope of the line,
as shown in the screenshot below.
Average data from
repeat trials
Excel was used to find any average values of the rate of oxygen
production (%/s) of all groups
11
Standard Deviation
Excel was used to find standard deviations of the rate of oxygen
production of all groups
Correlation
Excel was used to find R values
12
T Test
Graphpad was used to perform the t test and find the p and t values
(Finding P and T
values)
13
14
DISCUSSION, EVALUATION & CONCLUSION
Discussing and Reviewing (DCE Aspect 1)
The purpose of this lab was to investigate how different concentrations of hydrogen peroxide
(H2O2) affect the rate of enzymatic activity of catalase. By measuring the amount of oxygen
(ppm) given off over time (%/time) you could deduce the rate of oxygen production, a direct
reflection of the amount of hydrogen peroxide broken down by catalase, and thus the rate of
enzymatic activity. The data collected from the experiment showed that as the concentration of
the hydrogen peroxide increased, the rate of oxygen production increased, thus showing that
the rate of enzymatic activity increases.
This is shown by the collected data. The average rate of oxygen production (%/s) for 0.0%
H2O2 was -0.001050565. This is very low and the rate of oxygen production increases with
higher concentrations. The average for the 0.1% H2O2 is -7.76E-06 while the average for 0.5%
H2O2 is 0.00144371. The average for 1.0% H2O2 continues the trend as it averages at
0.00469768. The 3.0% H2O2 is the highest as it’s mean is 0.021205. Clearly, as the
concentration of hydrogen peroxide increases, the enzymatic activity of catalase increases,
causing it to break down more hydrogen peroxide, in turn causing an increase in the rate of
oxygen production. This is further supported by the graphical representation of the data (graph
2 mainly), which shows the correlation between the hydrogen peroxide concentrations and the
average rate of oxygen production. The R-value for the averages of the data set is
0.995508023, which indicates a strong positive correlation, meaning that as x increases (H 2O2
concentrations), y also increases (rate of oxygen production).
This is in accordance to the accepted data on catalase’s activity when decomposing
hydrogen peroxide. Although other scientists may not have achieved values that are exactly the
same, as there might have been too many alternative variables, they follow the same general
trend, an upward trend. As hydrogen peroxide values increase, the enzymatic activity also
increases. This occurs because as the amount of molecules in the concentrated surface area
increases, the molecules are able to collide more often with the activation site, causing more
product to be created, and thus an increase in enzymatic activity (Williams).
Evaluating Procedures and Suggesting Improvements: (DCE Aspect 2)
A) ACCURACY, PRECISION and RELIABILITY
The reliability of the data and procedure can be seen from the linear trend found in Graph 2 in
the data processing section. Each of the data points is very close to the line of best fit, meaning
the accuracy and precision of the raw data is acceptable. This is further shown through the R
value of the averages of the different substrate concentrations, 0.995508023, which shows
there is an extremely strong positive correlation, meaning the data points are very close to the
line showing an upward trend. There are however some slight inaccuracies in the data as
shown in Graph 3, which depicts error bars. The error bars are for the most part of acceptable
size, except those for the 3% hydrogen peroxide concentration and perhaps that for the 1%
hydrogen peroxide concentration. The 3% hydrogen peroxide error bar was larger than any
other error bar and seems to have such a large standard deviation that the variation in the data
may cause the averages and the data collected to lessen in precision. The 1% hydrogen
15
peroxide concentration does not seem to have such a large variation within the data, but in
comparison to the remaining three groups, it seems it does have a variation that hurts this
data’s precision. Although the reliability for these two groups may be slightly compromised, the
other groups have very low standard deviations, thus proving the data collected is fairly precise.
The accuracy of the experiment has also been proven to be quite high as the data follows the
accepted scientific information collected on enzymatic behavior. As hydrogen peroxide values
increase, the enzymatic activity also increases. This occurs because as the amount of
molecules in the concentrated surface area increases, the molecules are able to collide more
often with the activation site, causing more product to be created, and thus an increase in
enzymatic activity (Williams).
In my opinion, the overall precision and reliability is adequate as the data points do prove to be
fairly close to the linear trend line in Graph 2 and seem to be in accordance with each other and
the general upward trend. Although there might be areas where the accuracy and precision
lacks (mainly 3% hydrogen peroxide), it seems to be adequate for the majority of the groups
and doesn’t hurt the reliability of that data.
Precision
The precision of the data sets is fairly acceptable and yet some have a much higher amount of
variation than others. The 0.0% hydrogen peroxide has a standard deviation of 0.00078112,
0.1% hydrogen peroxide has 0.00043151, 0.5% hydrogen peroxide has 0.00122596, and 1.0%
hydrogen peroxide has 0.00237757. These are fairly low and are fairly close to each other,
although the 0.5% and 1.0% have higher standard deviations.
In comparison however, the 3.0% hydrogen peroxide solution, with a standard deviation of
0.00974628, had a much larger standard deviation. This might be because the higher
percentage of hydrogen peroxide caused more to be broken down, thus a speeding up of
enzymatic activity. Although this increase was observed for each of the groups, it was observed
at different severities as each group’s cube was cut differently. Some groups had bigger cubes
while others had smaller cubes and some cubes may have had different enzymatic
concentrations while others didn’t. While these inconsistencies in the different trials might not
have made a difference for the low concentrations of hydrogen peroxide as less enzymes may
have been able to be saturated, causing a more consistent result, it did as the concentrations
increased. This caused more substrate to bind with different enzymes, causing the enzyme
concentration and other factors to come into play. The surface area covered by hydrogen
peroxide might also have been a factor in this aspect. All of this together contributed to a higher
standard deviation (variety) within higher concentrations of hydrogen peroxide, particularly 3.0%
hydrogen peroxide. This may be improved by better controlling all variables other than the
independent variable and reducing random errors (specific improvements given below).
Systematic errors
As the data follows the accepted general trend, corroborated by Indiana University and John
Williams (described in depth above in Discussing and Reviewing and below in the Conclusion)
16
no real evidence points to real systematic errors. Therefore there are no observed systematic
errors.
Random errors
Although the experiment was fairly controlled, there were random errors that might have
occurred throughout the experiment. The following errors are listed in order of significance.
Random Error
Effect on experiment
Improvements
Some people cut the cube
smaller than others while some
cut the cubes bigger than
others as it was difficult to
accurately cut 1 cm3 liver cube.
If some people cut the cube
bigger than others, more
surface area would be available
for the catalase to decompose
the hydrogen peroxide. The
opposite would happen if the
cube was cut smaller than 1
cm3 as it would result in a
smaller surface area, leading to
less available catalase to
catalyse the reaction.
It might be better to use
better cutting equipment to
slice the cubes more
precisely. A more precise
and sharp scalpel may work
better in this case or an
electric cutter if they are
available for our use.
Each person may have spread
the hydrogen peroxide on the
cubes differently. Some may
have let the hydrogen peroxide
spread over more of the cube
while some might have spread
over less.
If more surface area is covered
by the hydrogen peroxide, more
enzymes might have been
saturated at once, increasing
the reaction rate. If less surface
area is covered, the opposite
happens and the reaction rate
decreases.
Ensure each group drops
the hydrogen peroxide in
the middle of the cube and
does not bump or touch the
oxygen chamber
afterwards.
People may have also put the
oxygen probe in the chamber
at different times, which may
have caused some of the
chambers to start out with
more oxygen and some with
less, as some oxygen may
have escaped before data
collection starts.
If oxygen escaped this may
have affected the rate of
oxygen production as it may
have caused the plateau to
near quicker, causing the rate to
decrease. This may not have
happened if less oxygen might
have escaped. This would
cause more variation in the data
and a higher standard deviation.
To ensure there is less
variety in the start time of
data collection, it would be
advantageous to ensure
everyone starts the timer 5
seconds after the cube was
lowered into the chamber
and the probe was put on.
This way there is no rush
for time and the starting
point for data collection is
fairly similar for each
substrate concentration.
Human reflex in timing the
process and pressing start
button in Logger Pro after the
oxygen probe was inserted.
People may have started the
data collection in Logger Pro
Starting data collection later or
sooner affects the rate of
oxygen production as the
starting value contributes to the
rate of reaction and the initial
180 seconds were said to be
It is difficult to lessen
human reflex as it is an
intrinsic fault, however it
might help if the person
pressing the start button
was not the person
17
slower or faster than others
(adds an uncertainty of ±0.2)
collected, not any later. This
would cause more variation in
the data and a higher standard
deviation.
inserting the oxygen probe
in the chamber, thus
ensuring he/she was fully
concentrated on the task.
There is always a possibility of
random issues with equipment
and people’s readings of them
(misreading due to parallax
error—for non-digital
equipment). It is also possible
that the calibrations were done
wrong by some people.
People may have added more
or less hydrogen peroxide if
they read the graduated
cylinder wrong or inaccurately.
This may have caused the
oxygen production to go up or
down as more or less substrate
my have been added (more
causes increase, less causes
decrease). If the calibrations
were done wrong, the
measurements carried out
could’ve increased or
decreased, depending on the
calibration mistake made.
Although we can never
know if one person has
made an error in their
readings, it would lessen
the possibility of large
errors if others checked
their measurements and
corroborated their values.
To ensure calibrations were
done correctly, every
individual must follow the
manual. It must also be
done carefully and as often
as needed in between
different trials.
B) EXPERIMENTAL WEAKNESSES AND LIMITATIONS AND IMPROVEMENTS
*The following limitations are listed in order of significance.
Improvement (to increase
Significance of the effect
accuracy and precision of raw
Limitations & Weaknesses
on accuracy &/or precision data, thus the reliability of the
trend observed)
Include values such as 5.0% and
Although it is difficult to
A lack of this may have
those closer to 10.0% or above if
acquire hydrogen peroxide at
caused a less accurate R
possible. In a secure lab in which
levels higher than 3.0%, it
value and a lack of
amounts are more controlled and
would be advantageous for
adequate data points to
better safety equipment is
the experiment to broaden its
make an acceptable
available this might be possible
spectrum of independent
conclusion.
(we might have been able to see
variables.
the plateau phase then).
Increase the number of trials and
Less data collected means the number of groups that are
a smaller sample size that
performing the experiment
Only ten trials were
in turn makes it difficult for (preferably as much as possible).
performed, which is not
us to evaluate the accuracy It might be better to use better
necessarily enough to make
and precision of the data. If cutting equipment to slice the
an accurate conclusion, as
we do not have a large
cubes more precisely. A more
the sample size is not large
enough sample size we
precise and sharp scalpel may
enough.
cannot make a valid
work better in this case or an
enough conclusion.
electric cutter if they are available
for our use.
18
Although the ruler seems to
be quite accurate, it might
lead to faulty readings, as it is
not digital.
There were some issues with
time management as we
might have rushed some of
the last trials in comparison to
the others.
Control of Variables
As each other the groups
performed their experiments on
varying days (3 different days
were used to fully carry out the
experiment by each class)
there are several things that we
might have failed to control.
These may have affected the
fairness and thus the validity of
the experiment (changes in the
dependant variable might not
have been reflection of solely
manipulations of the
independent variable)
This might cause the cube
size to either increase or
decrease, causing the
available surface area to
either increase or
decrease. An increased
surface area ensures an
increased amount of free
enzymes so the reaction
rate will increase. The
opposite will happen with a
decreased surface area
(smaller cube), causing a
decrease in reaction rate.
This could have brought on
more random errors and
could have caused slight
discrepancies in the
numbers, causing a greater
variation. This could’ve
affected the accuracy,
precision, and the
reproducibility.
Significance of the effect
on validity
All these factors may have
affected the precision and
increased variability in the
data.
A higher enzyme
concentration would have
Enzyme concentration and
ensured more available
size of the cube was perhaps
enzymes to catalyse the
different for each cube as
reaction, causing an
they were taken from different
increase in reaction rate. A
areas of the liver and were cut
lower enzymatic
different sizes (difficult to cut
concentration would
exactly 1 cm3)
therefore cause a
decrease in reaction rate.
19
An electronic cutter may prevent
this problem as it measures for
you (maybe be able to get readily
cut even slices in the store even).
A digital scale may be used to
measure the mass of the cubes
so as to ensure they are even
more similar to each other.
Ensure enough time is spent on
each trial and that we do not rush
the last trials due to fear of
running out of time. Ensure we
have all the time we need
(maybe 15 more minutes).
Improvement
These may be improved so as to
ensure a fairer test of the
variables, thus improving the
validity of the results.
It is difficult to ensure the enzyme
concentration is the same within
each of the cubes. Ensuring each
liver comes from a healthy
version of the same animal is as
much as we can do, but
regulating the cube size is
something we can improve on.
The pH level within each cube
may have varied, causing it to
affect the enzymatic rate of
reaction.
Although room temperature
should have been fairly
steady, fluctuations in
temperature could have still
affected the experiment
A larger cube creates the
same problem, as a larger
surface area is available,
causing more enzymes to
be available to catalyse the
reaction. A smaller cube
would therefore decrease
the reaction rate.
Diverging from the
optimum pH level, higher
or lower than 7, will cause
a decrease in enzymatic
activity and will therefore
cause a decrease in the
oxygen production over
time.
If the temperature were to
be higher on one day, the
enzymatic activity would
have increased. If it were
to be lower for other trials,
this might have slowed
enzymatic activity. Either
way, it would have caused
discrepancies in the data.
The optimum pH value of
catalase is 7, thus by using pH
buffers, we could ensure pH level
doesn’t interfere with the results
of the experiment.
Put each oxygen chamber and
the livers in an ice bath or a
container with regulated water
(room temperature: 37 degrees
Celsius). In any case,
temperature of the environment
should be recorded before and
after the start of each trial. Any
changes throughout, should be
recorded.
Although each weakness and limitation had an effect on the data, the most prominent issue
would have to be regulating the size of the cube. It was the most obvious factor as certain
cubes (0.1% in my case) were much smaller than others (3.0% in my case). If the cube has a
larger surface area, there are more enzymes available to catalyse the reaction, thus increasing
the reaction rate. If the cube has a smaller available surface area, there are less enzymes
available to catalyse the reaction, thus decreasing the reaction rate. This was a problem within
our lab as it was very difficult for us to cut the cubes into exactly 1 cm3.
As the method was fairly explicit and easy to follow, the reproducibility of the experimental
results should be fairly acceptable as long as similar conditions are kept. There is however a
large variability (standard deviation) for some of the hydrogen peroxide percentages, such as
3.0% and 1.0% (precision and accuracy not perfect). They might therefore be unable to get
exactly the same results, however they should get the same general trend as the trend was not
a weak trend, but a very strong positive trend, with a correlation between the averages of
substrate concentrations and the concentrations themselves of 0.995508023. This means that if
the experiment is done correctly, the scientist will most likely also collect data that forms some
kind of positive correlation. This positive correlation is also in accordance with the accepted
theory that if substrate concentration increases, the enzymatic activity also increases (described
in more detail in the conclusion and the Discussing and Reviewing section).
20
Concluding: (DCE Aspect 3)
The aim of the investigation was to determine the effect of substrate concentration (of
hydrogen peroxide) on the enzymatic activity of catalase. The data collected showed a direct
relationship between increase hydrogen peroxide concentrations and increased enzymatic
activity of catalase. This means that as the substrate concentration increased, so did the
enzymatic activity. Catalases reaction rate would increase with a higher concentration of
hydrogen peroxide and it would decrease with a lower concentration of hydrogen peroxide.
Thus we accept our experimental hypothesis and reject our null hypothesis.
The hypothesis was supported by evidence from collected data. As shown in the data
table, a hydrogen peroxide concentration of 0.0% rate of oxygen production of resulted in an
average of -0.00106%/s, a concentration of 0.1% resulted in an average rate of oxygen
production of -7.76E-06%/s, a concentration of 0.5% resulted in an average rate of oxygen
production of 0.00144371%/s, a concentration of 1.0% resulted in an average rate of oxygen
production of 0.00469768%/s, and a concentration of 3.0% resulted in an average rate of
oxygen production of 0.021205%/s. Graph 2 demonstrated that this set of collected data
resulted in a very strong positive correlation, with the correlation between the averages of the
substrate concentration and the substrate concentration being 0.995508023, a value very close
to 1. This shows that as the substrate concentration (hydrogen peroxide) increases, so does the
rate of oxygen production, thus the enzymatic activity.
The data supported the notion that as substrate concentration increases, enzymatic
activity increases until a maximum, when the enzyme is saturated, is reached. The substrate
binds with the enzyme through random collisions as each molecule is drifting through a solution.
Therefore the more concentrated the substrate is within a sol
ution, the more the
opportunity to collide with enzymes there will be. This will result in more conversion from
substrate to product and will increase enzymatic activity. There is however a limit as each
enzyme can only catalyse a reaction within a set amount of time, thus the enzyme reaches a
point of saturation (“Lab #4: Enzymes”, 3). In our experiment we observed the trend at the
beginning of the graph (increase), as we did not add a wide enough variety of substrate
concentration. It is likely that if we were to add a solution with a much higher percentage of
hydrogen peroxide than 3.0%, a saturation point will eventually be reached. The increase in the
first section of the graph is, however, in accordance with our data as there is a strong positive
correlation between the percentage of hydrogen peroxide in the solution and the amount of
oxygen produced (thus the enzymatic activity). Thus, as the substrate (hydrogen peroxide)
concentration increases, so does the enzymatic activity of catalase.
21
This figure shows the accepted trend described above. As substrate concentration is increase, the
enzymatic activity increases until saturation is reached (Figure from: “Lab #4: Enzymes”).
Works Cited
"Lab #4: Enzymes." Indiana University. Indiana University, n.d. Web. 5 Jan. 2014.
<http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab4.pdf>.
Williams, John. "The Decomposition Of Hydrogen Peroxide By Liver Catalase." NCBI. National Center
for Biotechnology Information, U.S. National Library of Medicine, n.d. Web. 11 Mar. 2014.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140981/>.
22
23