Biology Lab Report

The correlation between Acidity and Reaction Rate in an Enzyme-Substrate reaction

Catalase is an enzyme primarily found in the liver. It is used to catalyse the reaction of hydrogen
peroxide, into water and oxygen. Hydrogen peroxide enters the body through the consumption of fruits,
milk, honey and various food items containing pesticides (Grotz). There are even microbodies in the
body that produce hydrogen peroxide in the large intestine to regulate bacteria within the body.
Hydrogen peroxide, by itself, decomposes into water and oxygen over time.
2(𝐻2 𝑂2 ) → 2(𝐻2 𝑂) + 𝑂2
Catalase speeds up the decomposition exponentially by lowering the amount of activation energy
required for the reaction to take place. Without the enzyme the process could take up to half a day,
giving the hydrogen peroxide adequate time to demonstrate its corrosive properties within the body.
A factor affecting enzyme activity is pH value of the substrate. Different pH values force changes
in the shape of the active site of the enzyme, changing its ability to bind to the shape of its substrate
(ALevelNotes). The optimum pH level for the activity of catalase is a neutral acidity (pH of 7.0). However,
the acidity of a healthy body, on average, is regulated at slightly alkaline (pH of 7.4) and can vary
depending on the foods consumed and sickness (Chemcraft). People with acidity imbalances cannot
produce enough catalase to break down hydrogen peroxide into its non-lethal components, due to low
levels of enzyme activity, thus they are prescribed catalase supplements. However, too much catalase
will remove the all hydrogen peroxide and allow bacteria to thrive. Thus the aim of this investigation is
to attempt to find the reaction rate of catalase at various pH levels in order to calculate the potency of
catalase prescriptions for people with different body acidities.

Research Question
How does substrate solution acidity (pH value) affect the average reaction rate of an enzyme-catalysed
reaction, using catalase and hydrogen peroxide?

Hypothesis
It is predicted that the correlation between average reaction rate and the acidity of the substrate
solution will take the form of a normal distribution. This is because an increase or decrease in pH will
result in lower enzyme activity because of a change in the shape of the active site, resulting in lower
reaction rate. Natural selection would have ensured that some level of enzyme activity would exist for
most locations in the body where the enzyme is found alongside its substrate, as hydrogen peroxide
poses a health risk within the body and decreases chances of survival. The pH values, where catalase is
found, of a normal body range from 6.0-8.0 (Chemcraft), within which, it is predicted that catalase will
still be highly active. Based on this information it is predicted that the enzyme activity of catalase, with
pH as an independent and reaction rate as the dependent variable, can be modelled by a normal
distribution, with a maxima (mean) at pH 7.0 and a standard deviation of pH 1.0 (area between
6.0<x<8.0 is 68% of the total area under the graph).

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Independent Variable
The independent variable is the variable in which change is controlled to measure a change in the
dependent variable. The independent variable of this investigation is the acidity of the enzyme-substrate
solution. Using acidity buffers of different strengths, the pH levels of the solution will be set at 5.0, 6.0,
7.0, 8.0 and 9.0 for five trials with each value. The volume of pH buffer added will be equal to the
volume of the hydrogen peroxide solution, both of which will be fixed at 30ml per trial. This range is
centred at the optimal pH for enzyme activity and slightly exceeds the range of the body’s pH levels.

Dependent Variable
The variable in which change, relative to the change of the independent variable, being measured is the
reaction rate, taken as the change in substrate mass per second as an indicator of the decomposition
reaction progressing is the mass of hydrogen peroxide lost in the form of oxygen. An electronic balance
will be used to record the initial mass and the mass after 20 seconds, an interval where the reaction rate
is highest and the difference in mass is noticeable. The difference between these masses will be divided
by 20 to find the change in mass per second. New sets of chemicals will be used at the beginning of each
trial to prevent residue from previous trials affecting the data measurements taken.

Controlled Variables
To ensure ideal fair test conditions all the variables aside from the independent and dependent should
be kept the same to increase precision within a data set. However, this is not possible, thus an attempt
will be made to control the following variables that are most relevant to this experiment:
Solution Temperature (C)
Particle reactivity is higher as temperature is increased due to the speeding up of particles, increasing
the frequency of collisions and the reaction rate. In the case of enzymes, this applies till the point of
denaturing, when enzyme activity is reduced. To keep the reaction rate constant in each set of trials,
temperature will be kept at a constant throughout the trials. The solution temperature will be
monitored using a thermometer, and the experiment will only take place if temperature is between 23-
27 degrees Celsius.
Substrate and Enzyme Concentration
Raising substrate and enzyme concentration will increase the frequency of collisions between the
substrate and enzyme, increasing reaction rate. The concentration of the hydrogen peroxide substrate
will be fixed at 8% and the concentration of the catalase solution will be kept at 1%.
Volume of Enzyme Solution
A change in the volume of the enzyme solution means that there are more catalase molecules in the
solution to act as catalysts for the hydrogen peroxide decomposition, which would increase the reaction
rate by increasing the potential number of reactions that can happen per second. This will be kept at a
constant of 2ml per trial.
Volume of Substrate Solution
An increase in the volume of the substrate solution would, potentially, allow for more reactants to be
catalysed every instant by increasing the substrate molecules available for each enzyme molecule, thus
increasing the product yield and the recorded reaction rate. This will be controlled by using 30ml of
hydrogen peroxide every trial.
Volume of pH buffer
pH buffer is added to a solution to bring the solution close to a desired acidity. The volume added to a
solution determines how much the pH of the said solution changes by. Increasing this value in a trial
would change the pH of the said trial in relation to the rest, creating imprecision in the data set collected
by changing the enzyme activity of the catalase in the solution. This will be controlled by using 30ml of
pH buffer per trial.

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Materials
Material Quantity
Beaker (100ml) 5
Hydrogen Peroxide Solution (8%) 750ml
Catalase Solution (1%) 100ml
Electronic Balance (200g) 1
Syringe 1
Measuring Cylinder (50ml) 2
Thermometer 1
pH (5.0) Buffer 150ml
pH (6.0) Buffer 150ml
pH (7.0) Buffer 150ml
pH (8.0) Buffer 150ml
pH (9.0) Buffer 150ml
Stopwatch 1
Tissue Paper -

Safety precautions
Safety Equipment Quantity
Safety Glasses 1*
Laboratory Coat 1*
Disposal Bins As directed by supervisor
Tissue Paper -
Sink 1

*Note, these values are per person

 Safety glasses are advised when dealing with corrosive fluids such as hydrogen peroxide which
may cause irritation
 A laboratory coat is recommended to prevent the contents of the beakers from touching clothes
or bare skin in the case of an accident as the chemicals being used are corrosive
 The catalase, hydrogen peroxide and pH buffer solution should be disposed of in the sink unless
directed otherwise by a supervisor as the solution pH is relatively close to neutral and hydrogen
peroxide decomposes into oxygen and water over time, eliminating the risk of unwanted
chemicals forming.
 In the instance of damaged glassware, do not attempt to pick up with hands, move away and
wait for a supervisor or cleaning management to deal with the shards
 In the instance of spilled chemicals, the concentration of the chemicals being used are not very
high, causing minor irritation to the skin at max, thus, can be dealt with using a wet tissue paper
or left to cleaning management if the spillage exceeds what can be cleaned with tissue paper

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Method
1. Acquire equipment as presented in the Materials list and prepare safety equipment as per the
Safety Precautions list
2. Turn on an electronic balance (200g) and place a beaker (100ml) on top
3. Set the scale of the electronic balance to zero
4. Measure 30ml hydrogen peroxide in a measuring cylinder (50ml) and pour into said beaker
5. Measure 30ml pH (5.0) buffer in another measuring cylinder (50ml) and pour into said beaker
6. Measure temperature of solution in the said beaker using a thermometer, proceed to step 6 if
temperature is within 23-27 degrees Celsius
7. Use a syringe to inject 2ml of catalase solution (1% concentration) into the said beaker and
immediately start a stopwatch
8. Record the mass of the solution at approximately 0 seconds on an excel spread sheet
9. Record the mass of the solution at approximately 20 seconds on an excel spread sheet
10. Dispose of the chemicals within the beaker as directed by a supervisor
11. Wash the beaker and dry it
12. Repeat step 2-10 another 4 times using the same beaker, replacing the chemicals each time
13. Repeat step 2-11 another 4 times using different pH buffers (6.0; 7.0; 8.0; 9.0) and a new beaker
each time
14. Wash remaining glassware with water and leave them inside the sink
15. Wipe the surface of the electronic balance using a tissue paper
16. Calculate the reaction rate; calculate the change in mass per trial and divide each value by 20

Raw Data Table- The effect of changing pH on reaction rate in a catalase-hydrogen peroxide reaction
pH Change in mass of the solution; values taken at t=0 and t=20 (s) after start of reaction
Trial1 (g) Trial 2 (g) Trial 3 (g) Trial 4 (g) Trial 5 (g)
Initial Final Initial Final Initial Final Initial Final Initial Final
5 64.14 64.11 63.11 63.07 61.44 61.42 62.61 62.58 63.78 63.75
6 62.66 62.61 62.21 62.18 64.32 64.26 64.39 64.35 62.77 62.72
7 65.12 65.03 64.50 64.42 65.02 64.98 62.85 62.73 62.69 62.60
8 62.90 62.85 63.68 63.64 63.47 63.46 61.92 61.88 63.63 63.60
9 62.71 62.70 64.09 64.07 64.58 64.55 63.22 63.21 65.20 65.18

Observations
 Solution stayed transparent throughout the experiment
 Bubbles are formed in the solution immediately after catalase is added to the hydrogen
peroxide solution, but are attached to the beaker
 The hydrogen peroxide solution, prior to adding catalase, was releasing bubbles
 Rate of bubble formation was highest in the pH 7 solution and lowest on pH 5 and 9
 Initial masses of the solution in each trial are different
 The temperature of the solution throughout all the trials varied between 24 and 26 degrees
Celsius
 The decrease in mass was greatest in the first 5 seconds, after which, the decrease stagnated
 The pH buffer for the pH 9 set of trials was of a different chemical composition than the pH
buffers used in the rest of the experiment

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Calculations
Mean:
The most appropriate average to take in this investigation is the arithmetic mean, as it is not discrete
data that is being dealt with. A mean is required in order to minimize the effect of bias in the data when
presenting a conclusion, as experimental errors are random, affecting singular trials but not the whole
data set. Doing so will also result in one final value from a set of values, which can then be graphed or
manipulated. The formula for taking the mean is (where n is the number of data points in the set and x is
the point):
∑𝑛𝑟=1 𝑥𝑟
𝜇=
𝑛
Sample calculation from Processed Data Table
(−0.03) + (−0.04) + (−0.02) + (−0.03) + (−0.03)
𝑀𝑒𝑎𝑛 (𝑝𝐻 5) = = −0.03
5
Standard Deviation:
The standard deviation is a tool used to evaluate the precision of data within a set. It is useful for
determining outliers and anomalies, which can then be removed to make calculations more accurate
(refer to the mean calculations). An outlier in the data is a point that is outside 3 standard deviations
from the mean (outside 99.7% norm). The formula for standard deviation is:
𝑛
1
𝜎 = √ ∑(𝑥𝑟 − 𝜇)2
𝑛
𝑟=1

This calculation was conducted on an excel spreadsheet. A screenshot is below:

Standard Deviation to Mean Ratio:

The standard deviation to mean ratio is necessary to evaluate the relative precision within a data set.
When measuring larger quantities, it is expected that the standard deviation is higher. With this in mind,
the standard deviation to mean ratio is taken to evaluate the precision of the data set. A number above
40% is an indication of imprecision as it shows the values measured, range from 60-140% of the mean.
This value is acquire through the following formula.
𝜎
𝑅𝑎𝑡𝑖𝑜(%) = | | (100)
𝜇
Sample calculation from Processed Data Table#2
0.0071
𝑅𝑎𝑡𝑖𝑜(%) 𝑜𝑓 𝑝𝐻 5 = | | (100) = 23.5702 (2 𝑆𝑖𝑔. 𝐹𝑖𝑔. ) → 24
−0.03
Average Reaction Rate:
Reaction rate cannot be measured at an instant, thus average reaction rate is used to represent the rate
of reaction. The formula below calculates this, the mean in the graph below refers to the mean change
in mass.
𝜇
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑅𝑎𝑡𝑒 =
20
Sample calculation from Processed Data Table#3
−0.03
𝑅𝑎𝑡𝑒 𝑜𝑓 𝑝𝐻 5 = = −0.0015
20

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Processed Data Table- The effect of changing pH on reaction rate in a catalase-hydrogen peroxide
reaction
pH Change in mass of the solution; initial mass subtracted from final mass
Trial 1 (g) Trial 2 (g) Trial 3 (g) Trial 4 (g) Trial 5 (g)
5 -0.03 -0.04 -0.02 -0.03 -0.03
6 -0.05 -0.03 -0.06 -0.04 -0.05
7 -0.09 -0.08 -0.04 -0.12 -0.09
8 -0.05 -0.04 -0.01 -0.04 -0.03
9 -0.01 -0.02 -0.03 -0.01 -0.02

Processed Data Table#2- The effect of changing pH on reaction rate in a catalase-hydrogen peroxide
reaction
pH Mean change in mass of St.Dev. of data set (g) St.Dev. to mean ratio
the solution (g) (%)
5 -0.03 0.0071 24
6 -0.05 0.011 25
7 -0.08 0.029 34
8 -0.03 0.015 45
9 -0.02 0.0084 46

Processed Data Table#3- The effect of changing pH on reaction rate in a catalase-hydrogen peroxide
reaction
pH Mean Reaction Rate (gs-1)
5 -0.0015
6 -0.0023
7 -0.0042
8 -0.0017
9 -0.0009

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Graph

Correlation between pH and reaction rate in an

enzyme-catalysed reaction (catalase, H2O2)
Solution pH Value
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
-0.0005

-0.0009
-0.001

-0.0015 -0.0015
Reaction Rate (gs-1)

-0.0017

-0.002
-0.0023
-0.0025

-0.003

-0.0035

-0.004
-0.0042

-0.0045

Discussion
Post examination of the data showed a positive correlation between the pH value of the substrate
solution and enzyme activity, indicated by the increasing reaction rate, till pH 7, after which, there was a
negative correlation. In addition, the gradient between the pH 8 and pH 9 data point is less than the
gradient between the pH 7 the pH 8 data points, this trend is mirrored on the other side of the pH 7
point. This relation implies a normal distribution, where it is predicted that the gradient would plateau
at the x-axis as the pH increases or decreases from pH 7. In order to be able to accurately predict values
using a function, more data is required. In a normal distribution the modulus gradient after the maxima
should be equal to the modulus gradient before the maxima, making the curve symmetrical. This is not
the case as the reaction rate at pH 6 (-0.0023) does not equal the reaction rate at pH 8 (-0.0017), making
a skewed normal distribution. However, it is important to note that the recorded mass losses (processed
data table#2) of these points are within one standard deviation of each other. It is also important to
note that the standard deviation to mean ratio of the reaction rate at pH 8 and 9 are over 40%, implying
that there are discrepancies and outliers within the data set. However, this was expected, as the
measuring tool could only measure up to 2 decimal places of a gram, compromising accuracy. Because
of this, it is possible that the measured data, in part, is a misrepresentation of the actual relation
between the two variables.
As the relation between pH value of the substrate solution and enzyme activity take the form of
a skewed normal distribution with the maxima at pH 7, the hypothesis has been partially met. The
results disagree with the hypothesis as it was predicted that the correlation would take the shape of a
bell curve. In the graph, this is not the case as the data point at pH 8 is not equal to the data point at pH

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6. However, it is concluded that the hypothesis is ‘partially met’ and not ‘not met’ as there are
discrepancies in the data. The mean values for decrease in mass at pH 6 and pH 8 are within a standard
deviation of each other: -0.03+-0.1=-0.05+0.1 (processed data table#2).
The scientific reasoning behind this is firstly, that a change in pH changes the concentration of
hydrogen or hydroxide ions in a solution. This, to some extent, changes the shape of substrate,
especially in this case, as the substrate is a hydrogen-oxygen compound, making it unable to bind to the
active site of an enzyme molecule. Secondly, when the concentration of hydrogen is extremely high, a
change in protein structure can be observed, also changing the shape of the active site, making the
substrate unable to bind to the enzyme (Zipp-Park). Thirdly, hydrogen and hydroxide ions have an effect
on the ionization state of the components in amino acids (Brooklyn). In an enzyme, altering the ionic
bonds of amino acid chains changes the shape of the enzyme. This means that the enzyme specificity
would be altered and after a point, the enzyme would be rendered inactive or denatured. The reason for
the skew in the graph could be because hydroxide ions have more of an effect on the components in
catalase than hydrogen ions, altering the shape of the enzyme quicker.
After following the method, data was collected with over a 40% standard deviation to mean
ratio, implying that there were precision errors in the data. Based on the qualitative data observed,
there were several factors that could have influenced the data. Due to this, the validity of the method
needs to be addressed. Firstly, the oxygen bubbles released by the decomposition of the substrate
attached to the side of the beaker and were released at random intervals, interfering with the
measuring tool. Secondly, hydrogen peroxide is in a constant state of decomposition, meaning that the
substrate’s concentration is decreasing prior to adding the enzyme, lowering the frequency of collisions
between substrate and enzyme, thus lowering reaction rate. Thirdly, the apparatus used to measure the
decrease in mass of the enzyme-substrate solution could measure within 2 decimal places of a gram,
compromising accuracy of the data collected. Fourthly, the pH buffer for pH 9 was of a different
chemical composition than the other pH buffers, which could have interfered with the catalysing ability
of the substrate. The method however, was able to control several variables, such as solution
temperature through step 6, enzyme and substrate volumes, enzyme and substrate concentration and
pH value in each trial, creating some degree of precision. In addition, the results produced are
corroborated by the scientific explanation, as the shape of the graph resembles trend described, and the
point of maximum enzyme activity recorded is at pH 7, which is corroborated by other studies, implying
that the method is valid to some degree.

Evaluation
As there were several limitations in the method based on observations and data procured,
several improvements could be made to the method to improve precision and accuracy by controlling
more variables to measure consistent data that is close to what the actual values are. The following are
feasible improvements that can be made.
 Precision of the measuring tool
The measuring tool being used was an electronic balance. A limitation in the experiment was the
precision of the measuring tool as it could only measure to the nearest hundredth of a gram, whereas
the reaction was occurring on a scale smaller than this. This affected precision in the data set, rounding
values to the nearest hundredth. Within the timeframe of 20 seconds, for extreme pH values,
differences in the mass would have been occurring on the scale of thousandths of a gram. However, the
electronic balance was able to eliminate human error by presenting measurements digitally, eliminating
the necessity to read markings. This can be improved upon by using an electronic balance calibrated to
three decimal places of a gram. This would allow for better data collection, and a greater degree of
precision. A limitation of doing this is that there are more factors that would have to be addressed when
measuring in smaller units, such as vibrations in the ground or air, which can throw the data off.

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 Number of trials
A limitation with the current number of trials (five) is that the effect random errors have in the in the
data processing induces a large amount of bias when constructing an analysis. In a data set of five trials,
a random error would impact the precision of the data set far more than if there were fifty trials.
Although, the benefit of having five trials instead of a hundred is that changes in the environment over
time will have less of an effect on the data. If the number of trials were to be increased, it could be done
by allocating a set of trials to multiple groups (i.e. group A collects data for pH 7, group B collects data
for pH 8…) or by conducting multiple trials at the same time. This would require more material and
space, though, which may not be available.
 Range of values for the independent variable
A limitation with the range of values for the independent variable was that it was fixed between pH 5
and pH 9. While this is the relevant range for any enzyme activity within the human body, in order to
increase the accuracy of the trend line describing the relation between the independent and dependent
variables, it is necessary to increase this. As there is a finite range for pH, 0-14, it is possible to bring the
upper and lower limits of the range to a value close to a maximum and a minimum. However, this would
require more specialized equipment, as chemicals with extreme pH values are corrosive, and would
require a much greater degree of safety precautions to be taken. However, this can be accomplished
within a limited amount of time by increasing the number of lab personnel involved in the experiment
(i.e. assigning different pH values to each individual). This though, would increase the likelihood of an
accident in the laboratory.
 Interval between values of the independent variable
A limitation in the data would have been the limited interval between each value for the independent
variable. The result of using a limited number of values was a graph that had five data points. While five
data points is enough to establish an approximate trend, it is not enough to establish a conclusive
relation between two variables. Furthermore, introducing more values for the independent variable
would increase the accuracy of prediction by helping to establish a better model, as it is continuous data
that is being predicted. On the graph, it is indicated that pH 7 is the maximum point. However, this is not
the case as the optimum point is ‘approximately’ pH 7, the real optimum point could be anywhere in the
interval pH 6 and pH 8. Implementing this in the method would require more time and more resources.
However, conducting several experiments simultaneously with the aid of multiple individuals, each one
carrying out the procedure for a specific pH value.
 Exposure of hydrogen peroxide solution to environment
In the experiment, the exposure of hydrogen peroxide, prior to carrying out the method, to the
environment was a limitation, as hydrogen peroxide is constantly breaking down into water and oxygen.
Over time, there would be a decrease in concentration, which was one of the controlled variables
established. The method could be corrected by conducting multiple trials simultaneously, while each
beaker of hydrogen peroxide is of the same concentration. Doing this would require more equipment,
and a multiple personnel, each of which would complete a single trial, who would need to coordinate
their actions to carry out trials at the same time.

Conclusion
In conclusion, the hypothesis of this experiment was partially proven, as the relationship between
substrate pH and enzyme activity can be described using a skewed normal distribution with an optimum
point around pH 7, disproving the hypothesis. However, there were discrepancies in the data, which
indicate that the hypothesis may yet be correct, the standard deviation being one of them, which puts
the skewed values within range of each other. However, considering that there were several limitations
in the method (listed above) and that several improvements could have been made, the result of this
investigation is not conclusive information about the correlation between the independent and

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dependent variables. Further investigations that can be conducted are firstly, by correcting the method
and attempting this investigation again to attain a conclusive answer to the research question. Secondly,
an investigation could be conducted into the relation between pH level and the enzyme activity of other
enzymes within the human body, to see whether the same curve is produced; for this, the same method,
with improvements listed above would be used. Thirdly, the effect of temperature on enzymes over a
prolonged period should be observed so as to conduct an experiment into the effect temperature
change on the body, for people shifting countries or living in extreme conditions. Researching into these
topics will help doctors and physicians by furthering understanding on the effects of environmental
changes on the human body.

Works Cited

"Factor Affecting Enzyme Activity." A Level Notes. Ed. Sam Adam Day. N.p., n.d. Web. 29 Mar. 2016.
<http://alevelnotes.com/Factors-affecting-Enzyme-Activity/146?tree>.

Grotz, Walter. "Hydrogen Peroxide." Educate-Yourself. Educate-Yourself, n.d. Web. 29 Mar. 2016.
<http://educate-yourself.org/cn/hydrogenperoxidewaltergrotz20nov04.shtml>.

"PH and Enzymes." Biology Lecture. Brooklyn College, n.d. Web. 5 Apr. 2016.
<http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm>

"The Role of PH." Chemcraft. Acid PHree, n.d. Web. 29 Mar. 2016.
<http://www.chemcraft.net/acidph2.html>.

Zipp, Erik, and Chul-Won Park. "Effects of Temperature and PH on Enzyme Activity." Introduction to
Biochemical Engineering. RPI, n.d. Web. <http://www.rpi.edu/dept/chem-eng/Biotech-
Environ/Projects00/temph/enzyme.html>

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