Sarah Reyner

October 7, 2013
BSC 110L
Section 1
The Affects of pH on Peroxidase Enzyme

Abstract

Enzymes are proteins made up of amino acids that act as catalysts in chemical reactions.
The purpose of this experiment was to determine the optimum pH level that enables the highest
enzyme activity in a reaction. Our experiment consisted of measuring the initial reaction
velocity of reactions using peroxidase from a turnip as an enzyme. We conducted four different
trials using different pH levels. We hypothesized that a neutral pH would provide the highest
IRV. The results that we obtained in this investigation accepted this hypothesis because the pH
of 7.0 trial concluded with the highest IRV.
Introduction
Enzymes are proteins made up of amino acids that act as catalysts in chemical reactions.
A catalysts is a substance that speeds up a reaction without being consumed by the reaction.
Enzymes speed up reactions by lowering the amount of energy needed to start a reaction,
otherwise known as activation energy (Singh, N., & Singh, J. 2003).
Enzyme activity varies based on environmental factors such as pH and temperature as
well as concentration of substrate and products. In our experiment we will be investigating the
effect that pH has on chemical reactions and observing the optimal pH that enzymes perform at.
In order to asses enzyme activity we will be using the initial reaction velocity, or IRV, which
describes the speed of the reaction when the maximum amount of enzyme and substrate are
available (Welinder, K., & Mazza, G. 1975).
The amino acids that compose enzymes are arranged in a specific three-dimensional
shape, formally known as tertiary structure. The pH affects enzymes in regard to this tertiary
structure; they affect this shape and either enable or inhibit reactions. The pH affects the shape
of enzyme molecules by interfering with ionic bonds that are necessary to maintain their tertiary
structure (Singh, N., & Singh, J. 2003)

During this experiment we will be using the enzyme peroxidase from a turnip as a
catalyst and measuring its activity in varying pH levels. Peroxidase is an enzyme that breaks
down hydrogen peroxide into water and oxygen by transferring oxygen molecules from
hydrogen peroxide onto other organic compounds (Mazza, G., & Welinder, K. 1980). We will be
reacting this enzyme with the substrates guaicacol and hydrogen peroxide which will form water
and a brown colored compound called as tetraguaiacol as a product. Because pH alters enzyme
shape, we predicted that the enzyme would have the highest initial reaction velocity at a pH
around 7 because this is considered neutral which we predicted to leave the enzyme the least
denatured.
Procedure
The first step of the experiment was conducted as a class by the instructor.
Approximately 2 grams of turnip was cut from the inside and weighed on a scale. It was next
placed into a blender along with 200 ml of deionized water. After being blended, the mixture
was poured into a beaker though a coffee filter to rid any large turnip chunks. The resultant
mixture was the enzyme solution that was used in the preceding experiment.
The second step involved preparing and calibrating the calorimeter. A calorimeter
measures how much light passes through an object. We first prepared a blank by obtaining a
curvette and filling it with about two-thirds of deionized water. This blank was placed into the
calorimeter and used to calibrate the equipment.
Next, we prepared a solution for to use as a baseline. This solution was creating by first
adding 9.7 ml of deionized water, 0.1 ml of guaiacol, and 0.1 ml of hydrogen peroxide. We then
added 0.1 ml of the enzyme solution made from the turnip, covered it with dura film, and then

placed it into the calorimeter. We then recorded the calorimeters readings after every 15 seconds
for a total of 5 minutes or 300 seconds.
The next trials performed were done in a similar manner, but instead of using deionized
water, an equivalent amount of pH buffer was used. There were a total of 3 trials for a pH of 4.0;
7.0; and 8.0 that followed the same steps as the baseline trial. After the addition of the enzyme,
the solution was placed in the calorimeter and recorded every 15 seconds for 5 minutes.
Finally, we calculated the initial reaction velocity (IRV) for each of the trials by
measuring the slope for the data with the most significant discrepancy. The following formula
was used:
change in y
--------------- = IRV
change in x
Results
The baseline trial resulted in a steady increase of absorbency readings as each 15 second
interval progressed. The readings continued to increase all the way threw the final second, never
leveling off nor declining. The steepest increase was on the 225th and 240th second where it
jumped from 0.24 to 0.27. This 0.03 difference was used to calculate this trials IRV to be 0.002.
The second trial using a pH of 4.0 resulted with an initial decrease in readings followed
by a slow increase and slight leveling off at the end. The highest activity was observed in the first
15 seconds when the readings jumped from 0.08 to 0.02. These values where used to calculate
its IRV to be 0.00027.
The third trial using a pH of 7.0 displayed the highest activity due to its calculated IRV of
0.003. This was calculated in the first 15 seconds of the trial. The activity level of the
peroxidase increased progressively until the 150th second, and then leveled off and remained
0.11. The fourth trial was opposite of this trend, having a slight decrease at the initiation that was

quickly followed by a flatline of -0.04 after 60 seconds. The decrease from -0.05 to -0.04 was
used to calculate its IRV to be -0.0057.
According to these findings, the baseline trial portrayed the highest enzyme activity when
using the figures below as a source of reverence. However, when comparing initial reaction
velocities, the solution that used a pH of 7.0 measured the highest. Such measurements accepted
my hypothesis that predicted an neutral pH to be neutral.
Absorbency Readings for the Hydrogen Peroxide and Guaiacol Reaction in the Presence of
Peroxidase

Time
baseline
(second
s)

pH 4.0

pH 7.0

pH 8.0

0.04

0.08

15

0.04

0.02

0.05

-0.05

30

0.07

0.02

0.04

-0.05

45

0.08

0.01

0.05

-0.04

Time
baseline
(second
s)

pH 4.0

pH 7.0

pH 8.0

60

0.08

0.01

0.06

-0.04

75

0.09

0.02

0.07

-0.04

90

0.11

0.02

0.08

-0.04

105

0.12

0.02

0.09

-0.04

120

0.13

0.02

0.1

-0.04

135

0.15

0.03

0.11

-0.04

150

0.16

0.03

0.1

-0.04

165

0.17

0.03

0.11

-0.04

180

0.19

0.03

0.11

-0.04

195

0.2

0.04

0.11

-0.04

210

0.22

0.04

0.11

-0.04

225

0.24

0.04

0.11

-0.04

240

0.27

0.05

0.11

-0.04

255

0.29

0.05

0.11

-0.04

270

0.31

0.08

0.11

-0.04

285

0.32

0.11

0.11

-0.04

300

0.34

0.08

0.11

-0.04

Absorbency Readings for the Hydrogen Peroxide and Guaiacol Reaction in the Presence of
Peroxidase

baseline
0.002

pH 4.0
0.00027

pH 7.0
0.003

pH 8.0
-0.0057

Initial Reaction Velocities of the Hydrogen Peroxide/Guaiacol/Peroxidase

Reaction at Different pHs
Discussion
Among many other factors, pH plays a major role in determining the activity of enzymes.
All enzymes display a characteristic range of pH at which they are most active. This optimal pH
may be due to several factors involving the structure. Altering pH levels effect the shape of
enzymes (Mazza, G., & Welinder, K. 1980). This tendency to denature explains the results
observed in our experiment. According to our data, the neutral pH of 7 displayed the highest
activity level because it had the highest IRV than the other trials.
This experiment was hindered by several factors that potentially disqualified our findings.
Firstly, each trial was performed by a different set of students which prevented the assurance of
uniform procedure. Secondly, there was mechanical difficulty with some of the calorimeters.
These issues could be resolved by having the entire experiment performed by one group on one
calorimeter.
Resource Cited
Mazza, G., & Welinder, K. (1980). Covalent Structure of Turnip Peroxidase 7. European
Journal

Of Biochemistry, 108(2), 481-489.

Singh, N., & Singh, J. (2003). A Method for Large Scale Purification of Turnip Peroxidase and
Its Characterization. Preparative Biochemistry & Biotechnology, 33(2), 125.
Welinder, K., & Mazza, G. (1975). Similarities and Differences of Five Peroxidases from Turnip
and Horseradish. European Journal Of Biochemistry, 57(2), 415-424.