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Enzyme Activity Lab 13

AP Biology
(Peroxidase + Hydrogen Peroxide Complex Peroxidase + Water + Oxygen)
2H2O2 2H2O + O2 (gas)

Learning Objectives
The student is able to design a plan for
collecting data to show that all biological systems
are affected by complex biotic and abiotic
interactions (2D1 & SP 4.2, SP 7.2).
The student is able to use models to predict and
justify that changes in the subcomponents of a
biological polymer affect the functionality of the
molecule (4A1
& SP 6.1, SP 6.4).
The student is able to analyze data to identify
how molecular interactions affect structure and
function (4B1 & SP 5.1).

Basic background information

Basic protein structure


The concept of induced fit
The role of enzymes
That structure, function, and environment
are all required for maximal function of
enzymatic reactions

2H2O2 2H2O + O2 (gas)


Peroxidase is an enzyme that breaks
down peroxides, such as hydrogen
peroxide, and is produced by most cells in
their peroxisomes. Peroxide is a toxic
byproduct of aerobic metabolism. Various
factors abiotic and biotic could have
a major influence on the efficiency of this
reaction.

Label carefully
Gather all materials
Plan your procedure and know what you are
doing before you start. Do a mock run
There is timing involved, so be prepared to
start the timer as soon as you mix the
materials.
Make data charts ahead of time so you have
a place to put your results.

So, what is this lab about?


Turnip peroxidase is the enzyme that
catalyzes the reaction that breaks down
hydrogen peroxide into water and oxygen.

guaiacol
We can see the reaction because we are
adding guaiacol, an indicator of oxygen due to a
color change that occurs in its presence. The
more oxygen the deeper the brown the color
becomes. The compound guaiacol has a high
affinity for oxygen, and in solution,it binds
instantly with oxygen to form tetraguaiacol,
which is brownish in color. The greater the
amount of oxygen produced, the darker brown
the solution will become.

We can qualitatively or quantitatively


measure the color change after allowing the
reaction to occur.
A color palette is prepared by placing
different amounts of enzyme and substrate
mixture with distilled water so that the final
percent of the solutions varies by 10% in
each of the 11 test tubes prepared.
This will provide a way to view the different
colors that can be seen for the different
amounts of oxygen released at maximum
production. It will be used for comparison for
the other reactions.

Baseline is a universal term for most chemical


reactions. In this investigation, the term
is used to establish a standard for a reaction. Thus,
when manipulating components of a reaction (in
this case, substrate or enzyme) you have a
reference point to help understand what occurred
in the reaction. The baseline may vary with
different scenarios pertinent to the design of the
experiment, such as altering the environment in
which the reaction occurs. In this scenario, different
conditions can be compared, and the effects of
changing an environmental variable (e.g., pH)
can be determined.

Color palette

Rate can have more than one applicable


definition because this lab has two major options
of approach, i.e., using a color palette and/or a
spectrophotometer to measure percent of light
absorbance. When using a color palette to
compare the change in a reaction, you can infer
increase, decrease, or no change in the rate; this
inference is usually called the relative rate of the
reaction.
.

Part 1 Baseline materials+ labeling


1. E= enzyme the cold, turnip enzyme
label a 2.5 ml syringe to use to measure this.
2. P= product (oxygen) which is shown when
guaiacol reacts with it and turns brown
label a 2.5ml syringe to use to measure this
3. NB= buffer pH7 neutral buffer
label a 10ml syringe to use to measure this
4. S= substrate, Hydrogen peroxide
label a 2.5ml syringe to use to measure this
5. test tube- label SPNB substrate, product, neutral buffer
6. Test tube- label ENB enzyme, neutral buffer

Time
minutes

0 1

Scale/
number

B ENB
3ml NB neutral Buffer pH 7
1ml E= Enzyme turnip peroxidase

A SPNB
1ml NB neutral Buffer pH 7
1ml P = guaiacol
2ml Substrate hydrogen
peroxide

Cover with parafilm and mix . Use a disposable pipette


to transfer tube A to tube B. Cover and mix.
Immediately observe by comparing to the color palette and begin timing!
Observe every minute for 5 minutes.
Calculate the rate for the baseline. Color change/% oxygen over time

Time
minutes

0 1

Scale/
number

B ENB
3ml NB neutral Buffer pH 7
1ml E Enzyme turnip peroxidase

A SPNB
1ml NB neutral Buffer pH 7
1ml P = guaiacol
2ml Substrate hydrogen
peroxide
Immediately observe by comparing to the color palette and begin timing!
Observe every minute for 5 minutes.
Calculate the rate for the baseline. Color change/% oxygen over time

Part 2
This part will test how different pH levels will
alter the reaction rates as compared to the
baseline.
Gather your materials. Plan your procedure.
Put in the correct amounts. Be careful with the
buffers! Acids and bases can harm your skin
and eyes!
Label the test tubes,Set up your test tubes in
pairs.

Tubes 1,2,4,9,11,12
1 ml NB neutral buffer

1 ml P=product indicator
guaiacol
2 ml S= hydrogen peroxide

Tubes 3,5,6,7,8,10

Make a data
chart!

1 ml E=turnip peroxidase solution


3 ml buffer of the correct pH for the tube. For
example tube 3 use pH 3, tube 5 pH of 5 etc!

Tubes 1,2,4,9,11,12
1 ml NB neutral buffer

1 ml P=product indicator
guaiacol
2 ml S= hydrogen peroxide

Be ready!
Tubes 3,5,6,7,8,10 Mix tube 1 with tube 3.
Observe at time zero and
every minute for 5 minutes!
1 ml E=turnip peroxidase solution
3 ml buffer of the correct pH for the tube. For
example tube 3 use pH 3, tube 5 pH of 5 etc!

rate
Calculate the rate for each tube.
How much oxygen produced in 5 minutes?
Based on the readings over the 5 minute
time period compared to the color change
or % of oxygen.
% change over time= rate

graph
Rate for each pH

Assessment Questions

#3.
If you omitted the enzyme?
If you omitted the substrate?
If you omitted the indicator?
Based on your answer to #4 develop a
specific question to test for part 3 of the lab.
Your group must submit an experimental
plan for approval.

Part 3

Complete your experiment planning sheet


Record your data.
Data analysis
What conclusion can be drawn from your
groups data?
Create a presentation to share your
findings