Timothy Tovar

AP Biology
2nd Giannou
Enzyme Activity: How abiotic and biotic factors influence the rates of enzymatic
reactions.
II. Introduction
a. The purpose of the lab is to investigate the relationship between enzyme
structures and their function. It is also to make generalizations about
enzymes by studying just one enzyme, the factors that can change the rate
of enzymatic reaction speed, and which factors are biologically important to
enzyme activity.
b. When looking at enzymatic activity, it is important to know about what an
enzyme does. Enzymes important to biological systems. They are
responsible for speeding up chemical reactions in different systems. They are
able to do this by lowering the activation energy. Activation energy is the
energy needed for molecules to react with each other. The shape of the
enzyme is very important and thats how they determine their functions. Its
interesting to note that enzymes are very particular and selective (S153).
Within the enzymes, there are two different groups: there are catabolic
enzymes, and anabolic enzymes. Catalytic Enzymes, also named proteases,
which are found in many organisms. There are many examples of different
enzymes that can break down various substrate molecules. An example
would be a person taking a supplement of lactase, which is the enzyme that
breaks down lactose. The key factor for catalytic enzymes is the fact that
they hydrolyze large, complex molecules into simpler components (S153).
Anabolic enzymes are the other vital enzymes in living systems. ATP synthase
is an example of one. Their function is to restore energy in ATP by combining
ADP and a phosphate group. In this specific investigation, the enzyme
peroxidase will be obtained from a turnip. Peroxidase is an enzyme that

Timothy Tovar
AP Biology
2nd Giannou
breaks down peroxide, a product of aerobic respiration (S154). During the
investigation, its important to understand the different chemical reactions
that happen during the different procedures. In Procedure 1, when using
peroxidase, the general reactions are as follows:
Enzyme + Substrate Enzyme-Substrate Complex Enzyme + Product(s) +
G
Specifically for this reaction:
Peroxidase + Hydrogen Peroxide Complex Peroxidase + Water + Oxygen
2H2O2 2H2O + O2(Gas)(S155)
When looking at the reaction, the peroxidase is at the beginning and the end
of the reaction. This is because in every catalysts, the enzymes are not
consumed by the reactions. In this first procedure, the rate of enzymatic
reaction would be difficult to measure if there was not at least one substrate
to measure. In this reaction, the easiest molecule to measure would be
oxygen, which is a final product. This can be used by having an indicator.
The compound guaiacol has a high affinity for oxygen, and forms instantly
with oxygen to form tetraguaiacol. This forms a brownish color. The greater
amount of oxygen produced, the darker the solution (156).
c. Hypothesis:
Procedure 1
This procedure was trying to make a baseline. There wasnt a hypothesis
recorded, because the results would be the standard set for the reactions.
Procedure 2
pH 10 will be a lighter brown because there will be more acidity, and thus,
resulting in less oxygen produced in the final product.
pH 7 is a neutral acidity, resulting in the same color as the baseline that was
made.
pH 4 will be dark brown in color, because it is more basic. The more basic,
the more oxygen produced.
Procedure 3
As temperature increases, the rate of enzymatic activity increases to a
certain point. The point right before boiling might have a lot of reactions.

Timothy Tovar
AP Biology
2nd Giannou
However, the cold or room temperature will have the best effect because the
drop off temperature for human enzyme activity is around 40 C.
d. Variables:
Procedure 1
Independent Variable: Time that the enzyme would have to react with the
substrate.
Dependent Variable: The color of the reaction after certain time.
Control: This experiment does not have a control.
Constant: The type of peroxidase, the guaiacol, the amount of time, the type
of water, the amount of water, the type of peroxide, the amount of test tubes,
the amount of syringes, the amount of substance in each syringe.
Procedure 2
Independent Variable: The time that the enzyme has to react in the different
pH values.
Dependent Variable: The initial and ending color of the reaction after 5
minutes.
Control: The control is the baseline that was made in the first procedure.
Constant: The type of peroxidase, the guaiacol, the amount of time, the type
of water, the amount of water, the type of peroxide, the amount of test tubes,
the amount of syringes, the amount of substance in each syringe, the amount
of pH concentration for each buffer, the type of test tube, the
spectrophotometer.
Procedure 3
Independent Variable: The time the enzyme has to react in different
temperatures.
Dependent Variable: The initial and ending color of the reaction after 5
minutes.
Control: The baseline that was created in the first procedure as a reference.
Constant: The type of peroxidase, the guaiacol, the amount of time, the type
of water, the amount of water, the type of peroxide, the amount of test tubes,
the amount of syringes, the amount of substance in each syringe, the amount
of pH concentration for each buffer, the type of test tube, the
spectrophotometer, the hot plate, the type of ice, the thermometer, the type

Timothy Tovar
AP Biology
2nd Giannou
of tongs, the amount of syringes, the amount of thermometers, the amount
of water in each beaker, the temperature between the groups.
III. Methods
a. Materials
Procedure 1

Turnip Peroxide
.1% hydrogen peroxide
Guaiacol
Distilled Water
3 Test tubes and appropriate test tube rack
Timer
1,5,10 mL syringes

Procedure 2

Turnip Peroxide
.1% hydrogen peroxide
Guaiacol
Distilled Water
3 Test tubes and appropriate test tube rack
Timer
1,5,10 mL graduated pipettes, pipette pumps, or syringes,

spectrophotometer, 6 test tubes and appropriate test tube rack

Buffer with range of pH 4, 7, and 10

Procedure 3

b.

3 Thermometers
1 Heat Plate
3 500 mL beakers (Hot, Cold, Room Temp)
6 Test Tubes
1 Test Tube Rack
Tongs
Distilled Water
4 Syringes
Guaiacol
Peroxidase
H2O2 (Hydrogen Peroxide)
Procedures

Timothy Tovar
AP Biology
2nd Giannou
Procedure 1
Step 1: Using two 16x 150 mm test tubes, mark one substrate and the test tube
enzyme. To the substrate tube, add 7 mL of distilled water, .3mL of .1% hydrogen
peroxide, and .2 mL guaiacol for a total volume of 7.5 mL. Cover the test tube with a
lid and gently mix.
Step 2: To the enzyme tube, add 6.0 mL of distilled water and 1.5 mL of peroxidase
for a total volume of 7.5 mL. Cover the test tube with a lid and gently mix.
Step 3: Combine the contents of the two tubes (substrate and enzyme) in another
16x150 mL test tube, cover the tube with a lid, invert twice to mix, and place the
tube in a test tube rack. Immediately begin timing the reaction.
Step 4: Observe the color change for the next 5 minutes. Rotate the tube before
each reading. Record the observed color at 0, 1, 2, 3, 4, and 5 minutes.
Step 5: Use the color palette/chart (Figure 1) to help you quantify change in color
over time. Graph your data in your laboratory notebook.
Procedure 2
Step 1: Using clean 16x150 mL test tubes, make 3 sets of pairs of original substrate
and enzyme tubes for a total of 6 tubes or 3 pairs. This time you will substitute a
different pH buffer for the distilled water used in the original enzyme tube. Prepare
the tubes as follows and be sure to label them.
For each substrate tube in a pair, add 7 mL of distilled water, .3 mL of
hydrogen peroxide, add .2 mL of guaiacol for a total volume of 7.5 mL.
For each enzyme tube in the pair, add 6.0 mL of a specific pH solution and
1.5 mL of peroxidase for a total volume of 7.5 mL. For example, in the enzyme tube
of the first pair, you can substitute 6.0 mL of buffer solution of pH 3 for the distilled

Timothy Tovar
AP Biology
2nd Giannou
water; in the enzyme tube of the second pair, you can substitute 6.0 mL of buffer
solution of pH 5 for the distilled water, and so forth.
Step 2: Combine the substrate and enzyme tubes for all six pairs (total volume 15.0
mL per pair), cover with Parafilm, gently mix, and place the tubes back in the test
tube rack. Immediately begin timing the reactions.
Step 3: Record the observed color for each tube at 0 minutes and again at the time
you chose based on your results in Procedure 1. (Again, a cell phone and/or camera
are excellent ways to record color change.)
Step 4: Use the palette/color chart (Figure 1) to help you quantify the changes you
observe.
Graph your data as color intensity versus pH.
Procedure 3
-Set up the beakers at Hot, close to freezing, and room temperature.
- For hot= 95 Celsius or more, Freezing = 1 Celsius, Room = 19.5 Celsius. Put
thermometer in beaker and record data
- Set Up Enzyme. To the enzyme tube, add 6.0 mL of distilled water and 1.5 mL of
peroxidase for a total volume of 7.5 mL. Cover the test tube with a lid and gently
mix.
- Set up Substrate. To the substrate tube, add 7 mL of distilled water, .3mL of .1%
hydrogen peroxide, and .2 mL guaiacol for a total volume of 7.5 mL. Cover the test
tube with a lid and gently mix.
- Take the ENZYME Tube and change the temperature by placing in the different
beakers for (5 Minutes)
-Combine the substrate and enzyme and record the initial color. Record the final
color after 5 minutes. Use the color palette to determine the color.

Timothy Tovar
AP Biology
2nd Giannou
-Record group data and then average.
- Graph Data

IV. Data and Calculations

a. Data Tables
PROCEDURE 1
THE CHANGE OF COLOR VS. TIME TO MAKE BASELINE
Procedu
re 1
Time
Color
on pH

PROCEDURE 2
initial
color
pH 4

ph 7
2

Final
Color
pH 4

pH 7
9

pH 4
pH 7
ph10

ph 10
1
0

pH10
3

0 min
5 min
2
9
1
3
0
0

Timothy Tovar
AP Biology
2nd Giannou

PROCEDURE 3
Initial Color of the substrate and enzyme
mixture
Temp
1
Group 1
Group 2
Group3

Temp
2
1
1
0

Temp
3
1
1
0

1
1
2

Final Color of the Substate and Enyme Mixture

after 5 minutes
Temp
1
Group 1
Group 2
Group 3

1
1
0

Hot
Room
Cold

Average
Initial
1
1
1.33
Average
Final
1
5.3
5.67

Hot
Room

0 MIN
1
1

Hot
Room
Cold

Temp
2

5 MIN
1
5.33

Temp
3
6
5
5

5
6
6

Timothy Tovar
AP Biology
2nd Giannou
Cold

1.33

5.67

b. GRAPHS
PROCEDURE 1

Color of pH over a period of 5 Minutes

5
4
3

The Color of the pH 2

1
0

Time (Minutes)

PROCEDURE 2

Different pH Values Compared to a Change over Time

10
8
6

Different pH value

4
2
0
0 min

5 min

Intial and Final Time

pH 4

pH 7

ph10

Timothy Tovar
AP Biology
2nd Giannou

PROCEDURE 3

Different Levels of Enzymatic Reactions Differing on Temperature

6
5
4

Number on the Color Chart

3
2
1
0
1

Initial and Fnal Time (Min)

Hot

Room

Cold

c. Calculations
Procedure 2:
Y/X The slope of the different rates of change
Y is the change from initial to final color.
X is the total time recording (5 minutes).
Example: 7/5min= 7/5

V. Conclusion and Evaluation

a. Conluding
Procedure 1:
This procedure was making the baseline for the experiments later on. By
performing all of the procedures carefully, the darkest color recorded for the data
was 4. There wasnt too much change initially, however there were slight changes

Timothy Tovar
AP Biology
2nd Giannou
toward the end of the allotted time. When looking at the data, it seems as though it
was successful to create the baseline.
Procedure 2:
The different concentrations of the pH greatly affected the range of color in the
reaction when the peroxidase reacted with the substrate. The amount of oxygen
produced in the reaction reacted with the guaiacol and made it darker. Like I had in
my hypotheses, the more acidity in the concentration, the more the reaction the
enzyme has to go through. For example, in pH 4, the change in color was drastic
and almost instantaneous comparatively to pH 10. This was possible because of the
guaiacol reacting with the amount of oxygen produced in the reaction. The
peroxidase does not react with the oxygen to change the color, but instead, breaks
down the substrate into water, and oxygen.
Procedure 3:
When conducting the experiment, the group was uncertain on how to heat up the
enzyme. This was because we were unsure if the enzyme or the substrate should
go through the temperature. After discussing, the conclusion was to change the
temperature of the enzyme. After completing the proess to make the enzyme and
mixed it with the substrate, for 5 minutes we changed the temperature of the
enzyme and mixed it with the substrate. Almost instantly our hypothesis came true.
We hypothesized that the old temperature and the room temperature would see the
most enzymatic action, because the ideal temperature for a human enzyme to work
is 37 Celsius. If the temperature was too hot, like in one of the experiments, the
enzyme becomes denaturized. It is unable to function because of the condtions
around it. The human body is at the idea temperature for enzymes t work, so in
order to fully se the effects of different enzymatic reaction levels, the cold could be

Timothy Tovar
AP Biology
2nd Giannou
set much colder, the room temperature more like human body temperature, and the
hot anywhere about 37 Celsius.
b. Evaluating
Procedure 1: This entire experiment was pretty easy to follow. However, due to
inconsistency in my group, there was a few major problems. When we conducted
the experiment the first time, we had no reaction at all. The color never changed
from the clear color. I know this was a user error of some sort, because when we
tried it again, it worked.
Procedure 2: Going into the experiment, I remember being slightly confused about
what exactly we were experimenting with. But now that I have a better grasp of the
subject, I would have changed some things. The problems with my results werent
necessarily because of the experiment, but with the data collecting methods. There
was some discrepancy in the color analysis. People believed that 0 was the starting
number, while others believe it was 1. Regardless if those numbers seem
insignificant, it does change the slope and average.
Procedure 3: With this experiment, there were many things that could have been
different. Because we conducted our own experiment, and were not too sure about
some of the procedures, there was some steps that were different between the
groups. It was not anything major, but some people had different ideas on how to
conduct the experiment. The procedures were not too hard to follow because they
were the same as making a baseline.
c. Improving Investigation
Procedure 1: There was not too much to this experiment that could have been
messed up, however, out group managed to do it. The problem we encountered

Timothy Tovar
AP Biology
2nd Giannou
was the lack of reaction that occurred with the enzyme and substrate. This could
have been because of the lack of attention when applying the different compounds.
Procedure 2: The only problem that occurred with the Procedure 2 was the
discrepancy in analysis. The spectrophotometer was kind of blurry when looking at
the colors, and hard to tell exactly what level it was at. An easy improvement would
be a better chart to determine exactly what color it was.
Procedure 3: This experiment was all about what we thought would work. As a
small group, we determined what we should do, which caused some difference in
opinions. The data table was also hard to create. There were many that looked
different for each subgroup of 3. My data was like a three-way table, while others
were more like a two-way table. We collected all the same data, but it could have
been improved by recording color the color every minute for five minutes. The
chart looks like a line of best fit, because we collected 2 data points.

Timothy Tovar
AP Biology
2nd Giannou

Work Cited
AP Biology Investigative Labs; An Inquiry-Based Approach. New York: College Board, 2012.
S153-S159. Print.