EXPERIMENT 4 – Potato Polyphenol Oxidase (II)
BACKGROUND
We are now familiar with many aspects of the enzyme polyphenol oxidase and its reaction that
oxidizes catechol to benzoquinone. Here, we explore the substrate specificity of the enzyme,
determine its pH optimum for activity, and estimate the Michaelis-Menten constant. Instead of
using color estimation we will use a spectrophotometer to quantify the concentration of products
formed by this reaction.
PROCEDURE
Part A: Preparation of polyphenol oxidase extract (this method is the same as the one used in
the preceding experiment). We need to make the enzyme fresh today because proteases within
the lysate will degrade the polyphenol oxidase.
1. Set up a funnel lined with six layers of cheese cloth in a funnel rack placed over a 100 mL
beaker immersed in an ice bath.
2. Peel a small, or one-half of a large potato.
3. Dice potato and add to homogenizer. Add 50 mL of NaF solution. Note that NaF is a poison.
4. Blend in homogenizer for 1-2 min.
5. Pour homogenate through cheesecloth-lined funnel and collect soluble lysate in beaker. You
will need 20 mL of lysate for your experiment.
6. Wrap waste in paper towels and throw away.
7. Keep lysate on ice to minimize proteolysis of enzyme, and the production of colored products
within the lysate. Some particulate matter will settle to the bottom of the beaker. For your
enzyme studies only use the supernatant.
Part B: Effect of substrate type on enzymatic activity
1. Set up the experiment as shown in the Table 4 below. You may wish to stagger the time at
which you start the experiment so that you have time to perform all of the manipulations.
2. Prepare 37ºC water bath.
3. Prepare timer. Blank the spectrophotometer with water in a cuvette.
4. While the test tubes are still on ice, add 1 mL water to control test tube. Shake and record
color.
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5. Add 1 mL of substrates to other tubes (again you may wish to stagger the additions). Shake
and record colors.
6. Incubate all tubes for 5 min at 37ºC, transfer to cuvette and read absorbance at 430 nm
wavelength. Record color and color change.
7. Place back at 37ºC.
8. Repeat step 6.
9. Record data in Table 4.
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Table 4. Substrate specificity of potato poly phenoloxidase.
Test
Tube
#
Substrate
Enzyme
mL
Record Color Change
& A480 after 5 min at 370C
Mix
&
Incubate
@ 370C
A480
Color change
Mix
&
Incubate
@ 370C

1 mL
5 min
5 min
1 mL
catechol
1 mL
5 min
5 min
3
1 mL
phenol
1 mL
5 min
5 min
4
1 mL
hydroquinone
1 mL
5 min
5 min
2
A480
Color change

1 mL
water
(control)
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Record Color Change
& A480 after 10 min at 370C
Analysis of Data:
1. Record the A480 and color changes in Table 4 for the 5 and 10 minute incubations and submit this table in your lab report.
2. Does polyphenol oxidase exhibit absolute or group specificity? Define these terms to explain your answer.
3. For which substrate does polyphenol oxidase have the highest level of reactivity?
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Part C: Substrate saturation of enzyme
It is important to determine the kinetic parameters of enzymes. Two parameters that are key to
enzymes are the Km (Michaelis-Menten constant) and Vmax. The units of Km are typically
concentration and the units of Vmax are typically the amount of product produced per time. Km
value is the amount of substrate required for the enzyme to operate at one-half the maximum
velocity. A lower Km value often correlates with greater efficiency of enzyme catalysis. The
Vmax is the maximum rate of the reaction. The higher this value, the more quickly the enzyme
converts substrate into product.
1. Set up the experiment as shown in the Table 5 below. You will perform this reaction at room
temperature. The reactions will take place in the cuvette.
2. Prepare the test tubes with catechol and water.
3. Prepare the spectrophotometer for kinetic analysis at the proper wavelength and unit time.
Blank the spectrophotometer with water. Prepare parafilm strips for mixing cuvette contents.
4. Vortex the tubes.
5. Add the catechol/water solution to a cuvette. Add lysate, cover with parafilm, invert cuvette to
mix and read absorbance changes per unit time.
6. Evacuate cuvette contents, rinse with water, and repeat step 5.
7. Complete Table 5 below.
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Table 5. Catechol substrate saturation data.
Test
Tube
#
100 mM
catechol*
L)
Water
L)
Enzyme
L)
1
0
950
50**
2
10
940
50
3
20
930
50
4
40
910
50
5
60
890
50
6
80
870
50
7
120
830
50
8
180
770
50
9
240
710
50
10
300
650
50

Abs/min
(A480 /30 sec)
catechol
(mM)
** Start the kinetic reaction by adding lysate to a cuvette containing catechol and water;
immediately mix by inverting the parafilm-covered cuvette 3 times and record the
Abs/min.
Analysis of Data:
1. Calculate the m of catechol for each assay in Table 5.
2. With these data make a Michaelis-Menten graph. Plot Abs/min (V) on the y-axis versus mM
catechol ([S]) on the x-axis. Label your graph: Figure 1. Potato polyphenol oxidase substrate
saturation curve for catechol. Submit this figure in your lab report.
3. Estimate Km and Vmax from Figure 1 and submit these values with appropriate units in your
report.
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Part D: pH optimum of enzyme
The pH optimum of an enzyme can assist in finding the subcellular location of the enzyme. Enzymes
located in the cytosol tend to possess a pH optimum of approximately 7. Enzymes in the lysosomes,
where protein degradation reactions occur, possess a pH optimum of 5. To optimize enzyme activity
assays it is important to determine the pH optimum. Here you will find the pH optimum of polyphenol
oxidase.
1. Label test tubes 1-4.
2. Add appropriate buffer and catechol to each tube. Mix.
3. Prepare spectrophotometer.
4. Add solution to cuvette. Record color.
5. Add lysate to cuvette, cover with parafilm, invert three times, and read kinetic change. Return cuvette
contents to test tube. Incubate the test tube at 37ºC for 10 min.
6. Record color and color change at end of incubation period. Transfer contents to cuvette and record
absorbance.
7. Repeat steps 4-6 for remaining tubes.
8. Fill in Table 6 and include in your report.
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Table 6. Effect of pH on the activity of polyphenol oxidase.
Test
Tube
#
Volume
of pH
Buffer
(µL)
100 mM
catechol*
L)
Enzyme
(µL)


Mix

&
Abs/min Incubate
(A480 /30 sec) @ 370C
Record Final Color &
A480 after 10 min
at 370C
A480
1
650 L
pH 1.0
300
50 L**
10 min
2
650 L
pH 5.0
300
50 L
10 min
3
650 L
pH 6.0
300
50 L
10 min
4
650 L
pH 9.0
300
50 L
10 min
Color change
** Start the reaction by adding enzyme to a cuvette containing buffer and catechol; immediately mix
by inverting the parafilm-covered cuvette 3 times and record the Abs/min.
Analysis of Data:
1. Submit Table 6 in your lab report.
2. With these data, plot Abs/min (V) on the y-axis versus pH on the x-axis. You can place all four
plots on a single graph. Label your graph: Figure 2. Optimal pH for polyphenoloxidase.
Submit this figure in your lab report.
3. What is the optimal pH for potato polyphenoloxidase?
4. What are three possible ways in which pH can affect the formation of an ES complex? Illustrate
your three possibilities with a schematic equation and submit them in your lab report.
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