Laboratory 7 Report

Effects on Activity of the Peroxidase Enzyme

Jillian Taylor

Biology 1106

David Brake

21 October 2018
 A Study of the Peroxidase Enzyme: Lab 7

Abstract

Enzymes are a key component in the function of the metabolism, including reproduction,

interaction, and maintenance (Coleman, 2018). The enzyme studies in this experiment is the

peroxidase enzyme. 18). In the metabolic process, the toxic compound hydrogen peroxide (H​2​O​2​)

is released (Coleman, 2018). Peroxidase is the enzyme that removes H​2​O​2 before
​ it has the

chance to harm the cell (Coleman, 2018). This experiment studies the different factors that can

affect this specific enzyme and how that affects the rate of reactions in the metabolic process..

Varying levels of temperature, pH, and extract where tested in addition to an experiment

observing denaturation due to boiling. Data was placed in tables and graphed in scatterplots. The

experiments in boiling, extreme temperatures or pH, or varying extract levels had changes in the

reaction rate compared to the control. None of the variables studied had a 0 effect on enzyme

activity. Understanding the rate of reactions for enzymes is very important in science because

enzymes control, regulate, and catalyze an immense amount of reactions in nature. Without

enzymes, metabolic pathways would be “terribly congested” because the reactions occurring

would take an extensive amount of time (Reece, J.B., & Campbell). When an organism

experiences changes in temperature or pH, enzyme activity would be affected.

 Introduction

Metabolism is a highly important function in living organisms (Reece and Campbell,

2011). Enzymes play a vital role in the processes of the pathways within metabolism (Coleman,

2018). Some of these processes include digestion, gene expression, cellular respiration, cell

signaling, reproduction, intercellular interaction, and cell maintenance (Coleman, 2018).

Enzymes, specifically, are used as catalysts in metabolic reactions by lowering the activation

energy. (Reece and Campbell, 2011). They do this by bringing substrates closer together,

expediting the reaction process (Coleman, 2018). Many different conditions can affect the way

enzymes impact a reaction. Examples of these factors are the shape of an enzyme, temperature,

and pH (Coleman, 2018).

Peroxidase is an enzyme found in peroxisomes collected from a collection of plant tissues

(Coleman, 2018). In the metabolic process, the toxic compound H​2​O​2​ is released (Coleman,

2018). Peroxidase is the enzyme that removes H​2​O​2​ before it has the chance to harm the cell

(Coleman, 2018). Peroxidase speeds up the reaction between an oxidant and a reductant

(Hernández-Ruiz et. al., 2001). Because all of the reactants are colorless, guaiacol will be used to

represent one of the reagents in this lab (Coleman, 2018). Once it loses its hydrogen atoms, it

will turn a brownish color. A spectrophotometer will be used to measure the rate of change

(Coleman, 2018). This will provide the necessary data to determine how different factors affect

the peroxidase enzyme. Specifically, spectrophotometers record absorbance (Coleman, 2018).

Absorbance is a measure of how much of the substrate hydrolyzes in reaction (Coleman, 2018).
 The goal of this lab is to examine different factors and their effect on enzyme activity.

This lab begins with extracting the peroxidase enzyme from a turnip (Coleman, 2018). This is

important because peroxidase is the only enzyme that will react with H​2​O​2​, even though there are

many other enzymes present in plants (Coleman, 2018). Extraction of the enzyme from the turnip

must be conducted carefully, as factors like age and size can affect the peroxidase (Coleman,

2018). The overall goal of this is to prepare for future parts using the peroxidase enzyme

(Coleman, 2018). After this, the next part is important so as to standardize the dosages of the

enzyme. This is the control experiment for laboratory 7, with all future experimental data being

compared to the table made from this information (Coleman, 2018).

Experiment one tests the effect enzyme concentration has on the rate of reaction

(Coleman, 2018). It utilizes two concentrations to observe the change in absorbance. The

hypothesis is that the presence of more enzymes will catalyze the reaction. Experiment two

explores the effect initial pH has on the rate of enzymatic reaction using a constant peroxidase

enzyme concentration, but variable pH levels (Coleman, 2018). The hypothesis is that the higher

the pH, the longer the chemical reaction will take. It is predicted that the rate of absorbance will

decrease at high levels of pH. Experiment three investigates the effect boiling the enzymes has

on the rate of reaction (Coleman, 2018). The hypothesis is that after being boiled, the peroxidase

enzyme will make the reactions occur even faster. It is predicted that the rate of absorption will

increase when the extract is boiled. Experiment four explores the effect temperature has on the

rate of reaction (Coleman, 2018). This will be examined by using 4 different temperature of dry

baths to change the absorbance rate. The hypothesis is that the hotter the solution, the quicker the
enzymatic reaction rate will increase as the temperature of the solution is increased. It is

predicted that the solutions kept at warmer temperatures will have a higher rate of absorbance.

Materials and Methods

The following experiment was conducted at The University of Texas at Tyler in the

science laboratory at​ ​approximately 1 pm on October 15th, 2018. To prepare the materials, 8.0

grams (g) of peeled turnip tissue were weighed on a balance scale then mixed with 300 milliliters

(ml) of 0.1 molar (M) phosphate buffer at 4 degrees Celsius (°C) with a buffer solution at a pH

(BpH) of 7. When mixed, the two substances were blended rapidly for 20 seconds, then the

extract was filtered out using cheesecloth. Extracts were kept on ice while a disposable pipette

was collected. Said pipette was labeled “extract” (Coleman, 2018).

The next step of preparation was in collecting two test tubes labeling them as “Buffer pH

5” and “Buffer pH 7” then filled halfway with the respective stock solutions. In addition, two

disposable pipettes were labeled “Buffer pH 5” and “Buffer pH 7” to correspond with the test

tubes. Next, three more test tubes were obtained and labeled with the numbers 1-3 (Coleman,

2018). Test tube 1, the control, was filled with: 4.0 ml of 0.1 M phosphate buffer at pH 5, 1.0 ml

of 0.1 M phosphate buffer at pH 7, 2.0 ml H​2​O​2​, and 1.0 ml guaiacol to produce a total volume

of 8 ml in the tube. Test tube 2 was filled with: 1.0 ml of 0.1 M phosphate buffer at pH 5, 2.0 mL

H​2​O​2​, and 1.0 ml guaiacol to produce a total volume of 4.0 ml. Test tube 3 was filled with: 2.0

ml of 0.1 M phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate buffer at pH 7, and 1.0 ml

extract to produce a total volume of 4.0 ml (Coleman, 2018).

 After prepping the solutions, test tube 1 was poured into a cuvette and wiped with lens

paper. When handling, only the top 1/4th of the tube was touched. Using this solution, the

spectrophotometer was adjusted to zero absorbance at 500 nanometers (nm). After this, another

cuvette was wiped with a lens paper. The contents of test tube 2 were poured into test tube 3,

then quickly poured into the clean cuvette. The cuvette was then immediately placed in the

spectrophotometer. A timer was started at the same time and a reading was taken from the

spectrophotometer as the 0 reading. The absorbance was read at 20-second intervals and

recorded in Table 2. After 120 seconds, the tube was removed from the spectrophotometer and

the color change was noted (Coleman, 2018). The use of turnip extract produced a change of

around 0 to 1 in 120 seconds. (Coleman, 2018)​.

For the first experiment, five test tubes were collected and labeled 1-5. Tube 1 was filled

with the following liquids: 4.0 ml of 0.1 M phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate

buffer at pH 7, 2.0 ml H​2​O​2​,​ and 1.0 ml guaiacol at a total volume of 8.0 ml (Coleman, 2018).

Tube 2 was filled with: 1.0 ml of 0.1 M phosphate buffer pH 5, 2.0 ml H​2​O​2​, and ​1.0 ml

guaiacol to produce a total volume of 4.0 ml. Test tube 3 was filled with: 2.5 ml of 0.1 M

phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate buffer at pH 7, and 0.5 ml extract to

produce a total volume of 4.0 ml. Test tube 4 was filled with: 1.0 ml of 0.1 M phosphate buffer at

pH 5, 2.0 ml H​2​O​2​, a​ nd 1.0 ml guaiacol to produce a total volume of 4.0 ml. Test tube 5 was

filled with: 1.0 ml of 0.1 M phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate buffer at pH 7,

and 2.0 ml extract to produce a total volume of 4.0 ml (Coleman, 2018).

 Test tube 1 was poured into a cuvette and wiped with lens paper. Only the top ¼ of the

tube was handled. Using the substance from tube 1, the spectrophotometer was adjusted to zero

absorbance at 500 nm. Next, the contents of tube 2 and 3 were mixed and poured into a cuvette.

Measurements were collected at 15 second intervals for 60 seconds and recorded in Table 5.

Next, the contents of tubes 4 and 5 were combined and poured into a cuvette. Measurements

were taken at 15 second intervals for 60 seconds and recorded in Table 5 (Coleman, 2018).

For the second experiment, 9 test tubes were collected and labeled 1-9 (Coleman, 2018).

Test tube 1 was filled with: 6.0 ml of 0.1 M phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate

extract, and 1.0 ml guaiacol to produce a total volume of 8.0 ml (Coleman, 2018). Test tube 2

was filled with: 2.0 ml H​2​O​2​, and 1.0 ml guaiacol to produce a total volume of 3.0 ml. Test tube

3 was filled with: 4.0 ml of 0.1 M phosphate pH 3 and 1.0 ml extract to produce a total volume

of 5.0 ml. Test tube 4 was filled with: 2.0 ml H​2​O​2​, and 1.0 ml guaiacol to produce a total

volume of 3.0 ml. Test tube 5 was filled with: 3.0 ml of 0.1 M phosphate buffer at pH 5 and 1.0

ml extract to produce a total volume of 5.0 ml. Test tube 6.0 was filled with: 2.0 ml H​2​O​2​, and

1.0 ml guaiacol to produce a total volume of 5.0 ml. Test tube 7 was filled with: 4.0 ml of 0.1 M

phosphate buffer at pH 7 and 1.0 ml extract to produce a total volume of 5.0 ml. Test tube 8 was

filled with: 2.0 ml H​2​O​2​, and 1.0 ml guaiacol to produce a total volume of 3.0 ml. Finally, test

tube 9 was filled with: 4.0 ml of 0.1 M phosphate buffer at pH 9.0 and 1.0 ml extract to produce

a total volume of 5.0 ml (Coleman, 2018).

Tube 1 was poured into the cuvette, wiped clean, then used to adjust the

spectrophotometer to zero absorbance at 500 nm. Next, tubes 2 and 3 were combined into a
cuvette and measured at 15 second intervals for 60 seconds. The same test was conducted for

tubes 4 and 5, 6 and 7, and 8 and 9 (Coleman, 2018). All collected data was recorded in Table 7.

For the third experiment, a test tube was obtained, filled with 3 mL extract, then placed in

a hot dry bath. 5 minutes later, the tube was removed and allowed to cool to room temperature. 3

more test tubes were gathered and labeled 1-3 (Coleman, 2018). Test tube 1 was filled with: 5.0

ml of 0.1 M phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate buffer at pH 7, 1.0 ml extract,

and 1.0 ml guaiacol to produce a total volume of 8.0 ml. Test tube 2 was filled with: 1.0 ml of

0.1 M phosphate buffer at pH 7, 2.0 ml H​2​O​2​, and 1.0 ml guaiacol to produce a total volume of

4.0 ml. Test tube 3 was filled with 1.0 ml of 0.1 M phosphate buffer at pH 5, 1.0 ml of 0.1 M

phosphate buffer at pH 7, and 2.0 ml extract to produce a total volume of 4.0 ml (Coleman,

2018).

The contents of test tube 1 were emptied into a cuvette, wiped clean, then again used to

adjust the spectrophotometer to zero absorbance at 500 nm. Next, the contents of 2 and 3 were

mixed then poured into a clean cuvette. The absorbance was measured at 15 second intervals for

60 seconds and recorded in Table 9 (Coleman, 2018).

For the fourth experiment, 9 test tubes were obtained and labeled 1-9. Tube 1 was filled

with: 6.0 ml of 0.1 M phosphate buffer at pH 5, 1.0 ml extract, and 1.0 ml guaiacol to produce a

total volume of 8.0 ml. Test tube 2 was filled with: 1.0 ml of 0.1 M phosphate buffer at pH 5, 2.0

ml H​2​O​2​, and 1.0 ml guaiacol to produce a total volume of 4.0 ml. Test tube 3 was filled with:

2.0 ml of 0.1 M phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate buffer at pH 7, and 1.0 ml

extract to produce a total volume of 4.0 ml. Test tube 4 was filled with: 1.0 ml of 0.1 M

phosphate buffer at pH 5, 2.0 ml H​2​O​2​, and 1.0 ml guaiacol to produce a total volume of 4.0 ml.
Test tube 5 was filled with: 2.0 ml of 0.1 M phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate

buffer at pH 7, and 1.0 ml extract to produce a total volume of 4.0 ml. Test tube 6 was filled

with: 1.0 ml of 0.1 M phosphate buffer at pH 5, 2.0 ml H​2​O​2​, and 1.0 ml guaiacol to produce a

total volume of 4.0 ml. Test tube 7 was filled with: 2.0 ml of 0.1 M phosphate buffer at pH 5, 1.0

ml of 0.1 M phosphate buffer at pH 7, and 1.0 ml extract to produce a total volume of 4.0 ml.

Test tube 8 was filled with 1.0 ml of 0.1 M phosphate buffer at pH 5, 2.0 ml H​2​O​2​, and 1.0 ml

guaiacol to produce a total volume of 4.0 ml. Test tube 9 was filled with: 2.0 ml of 0.1 M

phosphate buffer at pH 5, 1.0 ml of 0.1 M phosphate buffer at pH 7, and 1.0 ml extract to

produce a total volume of 4.0 ml (Coleman, 2018).

Tubes 2 and 3 were incubated in an ice bath at approximately 4°C. Tubes 4 and 5 were

kept at room temperature at 23°C. Tubes 6 and 7 were heated to 32°C. Tubes 8 and 9 were

heated to 48°C. All test tubes were allowed to adjust to their environments for 15 minutes until

they reached equilibrium. Test tube 1 was again used to adjust the spectrophotometer using the

same technique as before. Next, tubes 2 and 3, 4 and 5, 6 and 7, and 8 and 9 were mixed with

their respective pairs. Measurements were taken every 15 seconds for 60 seconds and recorded in

Table 11. Tubes were taken out of their temperature conditions right before being tested

(Coleman, 2018).
 Results

Table 1: Control Experiment

Time (seconds) Absorbance at 500 (nm)
0 0.611
20 1.544
40 2.235
60 2.705
80 2.898
100 3.23
120 3.22

Figure 4: Control Experiment

Production of tetraguaiacol in the conversion of H​2​O​2​ into H​2​O by peroxidase

Figure 1 shows the results of the first experiment in the enzyme lab, the control. This

experiment sets a base level for future data to be compared to. Due to machine error, the initial

absorbance fell at 0.611 nm. The slope of absorbance rate for the line of best fit in the control
experiment was calculated by change-in-y divided by change-in-x. For this experiment, that

calculation yielded a slope of 0.96.

Table 2: ​Varying Amount of Extract Experiment

Time
(seconds) Control (nm) .5 mL extract (nm) 2.0 mL extract (nm)
0 0.611 0.548 1.966
15 N/A 0.499 2.997
30 N/A 0.425 3.540
45 N/A 0.355 3.850
60 2.705 0.293 3.310

Figure 2: Varying Amount of Extract Experiment

Production of tetraguaiacol in the conversion of H​2​O​2​ into H​2​O by peroxidase for
different amounts of extract

Figure 2 shows the results of the experiment testing changes in the amount of extract on

the production of tetraguaiacol. Shown in figure 1, the control experiment with 1.0 mL of extract

had a slope of 0.96. The line of best fit for the 2.0 mL extract sample is steeper than the control
(Figure 2). The line for the 0.5 mL extract is less steep than the control and goes in the negative

direction (Figure 2). The table contains missing data because the control experiment did not use

the same intervals as the rest of the experiments (Table 2).

Table 3: ​Varying pH Experiment

Time (seconds) Control (nm) pH 3 (nm) pH 5 (nm) pH 7 (nm) pH 9 (nm)

0 0.611 0.268 0.755 0.986 0.949
15 N/A 0.247 0.987 1.529 1.235
30 N/A 0.235 1.034 1.801 1.677
45 N/A 0.215 1.076 1.923 2.040
60 2.705 0.200 1.121 1.946 2.280

Figure 3: Varying pH Experiment

Production of tetraguaiacol in the conversion of H​2​O​2​ into H​2​O by peroxidase at different pHs
 Figure 3 shows the results of the experiment testing the effects of differing pH on the

production of tetraguaiacol. The blue line represents the control experiment, which used 3 mL

pH 5 and 1.0 mL pH 7. The slope of the line of best fit is steeper than any of the other lines

(Figure 3). The experiment using pH 9 had the reaction most similar to the control (Figure 3).

The reaction using pH 3 had the most different absorbance rate, with the slope going in the

negative direction (Figure 3). The reactions at different pHs look different than the control

because they had a slower rate of reaction. The table contains missing data because the control

experiment did not use the same intervals as the rest of the experiments (Table 3).

Table 4: ​Denaturalization Experiment

Time (seconds) Control (nm) After boiling (nm)

0 0.611 0.333
15 N/A 0.245
30 N/A 0.215
45 N/A 0.200
60 2.705 0.179

Figure 4: Denaturalization Experiment

Production of tetraguaiacol in the conversion of H​2​O​2​ into H​2​O by peroxidase after boiling the
extract
 Figure 4 shows the results of the production of tetraguaiacol after boiling the extract.

Compared to the control experiment, the production rate was very different after boiling (Figure

4). The slope after boiling goes in the negative direction (Figure 4). The table contains missing

data because the control experiment did not use the same intervals as the rest of the experiments

(Table 4).

Table 5: ​Varying Temperatures Experiment

Time
(seconds) Control (nm) 4°C (nm) 23°C (nm) 32°C (nm) 48°C (nm)
0 0.611 0.087 0.412 0.295 0.602
15 N/A 0.071 0.791 0.855 0.864
30 N/A 0.142 1.220 1.216 0.913
45 N/A 0.241 1.490 1.466 0.862
60 2.705 0.342 1.611 1.614 0.791

Figure 5: Varying Temperatures Experiment

Production of tetraguaiacol in the conversion of H​2​O​2​ into H​2​O by peroxidase at different
temperatures
 Figure 5 shows the production of tetraguaiacol after the extract was set to different

temperatures. The slope of the control experiment was the steepest the slope of the reactions at

32°C and 23°C were most similar to the control (Figure 5). The reactions at different

temperatures look different than the control because they had a slower rate of reaction. The table

contains missing data because the control experiment did not use the same intervals as the rest of

the experiments (Table 5).

Discussion

The experiments observe oxygen (O2) production, the starting rate of H2O2

decomposing activity, and peroxidase inactivation and the determination of its lasting effect on

activity (Hernández-Ruiz et. al., 2001). The results show a relationship between the reactions

between an oxidant and a reductant and concentration of the horseradish peroxidase enzyme

(Hernández-Ruiz et. al., 2001).

Experiment one tested the effect the amount of extract had on the production of

tetraguaiacol. Peroxidase speeds up the reaction between an oxidant and a reductant

(Hernández-Ruiz et. al., 2001). Because of this, it was hypothesized that the presence of more

turnip extract will increase the rate of absorption. The experiment produced the predicted result,

with the 2.0 mL extract sample reacting at a much faster rate than the 0.5 mL sample (Figure 1).

In fact, the 0.5 mL extract actually went in the opposite direction.

Experiment two tested the effect of different pH on the production of tetraguaiacol by

peroxidase. Originally, it was hypothesized that a higher pH would cause a lower rate of

absorbance. High and low pHs have an effect on enzyme activity, with the extreme values
resulting in complete failure (Introduction to Enzymes). The experiment did not yield results that

align with my hypothesis or with online research. The higher the pH, the quicker the enzyme

reacted.

Experiment three tested the effect of boiling the extract in the production on tetraguaiacol

by the peroxidase enzyme. The hypothesized outcome is that boiling the extract will cause the

rate of absorbance to increase. The data did not support this hypothesis. The data collected shows

that after being boiled, the enzyme no longer worked. The slope of the line actually went in the

negative direction, but this may have been due to machine error. No absorption occurred after the

enzyme was boiled due to denaturation. Denaturation occurs at extremely high temperatures

when molecular bonds are broken down (Enzymes).

Experiment four observed the effects of temperature on the production of tetraguaiacol.

Initially, it was hypothesized that higher temperatures will result in a higher rate of absorbance.

The resulting data showed that this predicted outcome was incorrect. The trendlines for the

lowest and highest temperatures showed that very little reaction occurred. This is because

enzymes have an optimal temperature at which the rate of absorption is the highest. For human

enzymes, the optimal temperature is typically around 35–40°C (Reece, J. B., & Campbell).

In experiment 1, the problems were most likely due to machine error. When calibrated,

the machine was not able to reach 0 due to malfunction. For future experiments, the

spectrophotometer will need to be properly calibrated prior to conducting the experiment.

In experiment 2, my results contradict the theory that enzymes operate best under a certain

threshold (Introduction to Enzymes). Other investigations do not relate to my results. This may

have been a result of miscalibration of the machine. To fix a majority of the problems in this lab,
future experimenters need to be careful that their equipment is properly maintained.

Understanding the rate of reactions for enzymes is very important in science. Enzymes

control, regulate, and catalyze an immense amount of reactions in nature. Without enzymes,

metabolic pathways would be “terribly congested” because the reactions occurring would take an

extensive amount of time (Reece, J.B., & Campbell). When an organism experiences changes in

temperature or pH, enzyme activity would be affected. This is true because of the observed data

in this lab manual. Enzymes require optimal conditions to operate (Reece, J.B., & Campbell).

Therefore, overheating/overcooling or abnormal pH will cause a malfunction in metabolic

processes.
 Literature Cited

Bagger, S. and Williams, R. J. P. (1971) Intermediates in the reaction between hydrogen

peroxide and horseradish peroxidase. Acta Chem. Scand. 25, 976–982

Coleman, J. L. (2018) Cell Properties and Processes. Biology 1106 Laboratory Manual. Pages

75-87

Gajhede, M., Schuller, D. J., Henriksen, A., Smith, A. T. and Poulos, T. L. (1997) Crystal

structure of horseradish peroxidase C at 2.15 A/ resolution. Nat. Struct. Biol. 4,

1032–1038

Hernández-Ruiz, J., Arnao, M. B., Hiner, A. N., García-Cánovas, F., & Acosta, M. (2001).

Catalase-like activity of horseradish peroxidase: Relationship to enzyme inactivation by

H​2​O​2​. Biochemical Journal, 354(1), 107-114. doi:10.1042/0264-6021:3540107

Reece, J. B., & Campbell, N. A. (2011). Campbell biology. Boston: Benjamin Cummings /

Pearson. Pages 9-11

Introduction to Enzymes. (n.d.). Retrieved November 12, 2018, from

http://www.worthington-biochem.com/introbiochem/effectsph.htm​l

Enzymes. (n.d.). Retrieved November 12, 2018, from

http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.html