Kinetic Analysis of Catechol Oxidation by Catechol Oxidase from Apples
Jason Freischlag
2/29/14
Chem 403 Dr. Porter
Abstract
The purpose of this experiment was to use rates of reactions to determine which apple would
make the best fruit salad. This is possible by examining the oxidation of catechol which is
responsible for the browning of apples. Catechol oxidase was used to accelerate the oxidation of
catechol into 1,2-benzoquinone. The Vmax for granny smith was found to be 2.78 x 10-8 (M/s), the Vmax for
red delicious was found to be 3.95 x 10-8 (M/s). The Vmax for golden delicious was found to be 5.59 x 10-8
(M/s). The Kmax for granny smith was 0.0007386 M, The Kmax for red delicious was 0.0009988 M, and the
The Kmax for golden delicious was 0.0055163 M. This data suggests that granny smith apples have the
lowest maximum rate of reaction and therefore would be the best apple for a fruit salad.
Introduction
Quinones are a class of organic compounds that are defined by their aromatic structure and
oxygen functional groups as can be seen in figure 1.
Figure 1. 1,4-benzoquinone, or simply quinone
Quinones are produced in apples only when the apple is damaged or the skin is removed and the
inside is exposed to oxygen. It is this characteristic of quinones that have led many to believe they
may have antibiotic properties.1
Catechol Oxidase is an enzyme that accelerates the reaction between oxygen and
hydroxylated benzenes to produce quinones and water.2 The oxidation of catechol, a hydroxylated
benzene, to form 1,2-benzoquinone and water will be used to examine the effect of enzyme
concentration on reaction rate. This oxidation reaction can be seen in figure 2.
Figure 2. Oxidation of Catechol to 1,2-benzoquinone
Enzyme catalyzed reactions can be described using the Michaelis-Menten mechanism seen
in equation 1.
𝐸 + 𝑆 ↔ [𝐸𝑆] → 𝐸 + 𝑃
In this experiment the enzyme, E, is catechol oxidase from various apples; the substrate, S, is
catechol; and the product, P, is 1,2-benzoquinone. This relationship will be used to study the
1)
relationship between reaction rate and concentration by using ultraviolet-visible spectroscopy to
determine concentration from absorbance at 540 nm where only quinone absorbs light.
Experimental
A stock solution of the substrate was created with catechol in water at a concentration of 500
mg/L. Catechol oxidase solutions were prepared by peeling and coring a granny smith apple, a
golden delicious apple, and a red delicious apple separately. The flesh from each apple was blended
with ice water and the juice was extracted, equipped to bubble nitrogen, and stored on ice to prevent
premature browning. Samples were created by mixing 0.50 mL of catechol oxidase solution with a
range of substrate catechol solution decreasing from 2mL to 0mL in equal portions. Each of these
nine solutions was then diluted to 2.5 mL and absorbance vs. time spectra were collected using a
Vernier Lab Quest Spectrometer. The spectrometer was set to measure absorbance at 540 nm
every 1 second for 400 seconds. A tenth solution was created using no enzyme, 2 mL of substrate,
and 0.50 mL water which represents the rate of the reaction with no enzyme present. Trials 9 and 10
were used to standardize the results by subtracting 9 and 10 from each other trial. Table 1 shows
the makeup of each solution in table form.
Table 1. Composition of each solution
Trial
1
2
3
4
5
6
7
8
9
10
Vol E
(mL)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0
Vol S
(mL)
2
1.75
1.5
1.25
1
0.75
0.5
0.25
0
2
Vol H2O
(mL)
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
0.5
Results and Discussion
Beer’s law, found in equation 1, is useful for determining the concentration of a substance
given the absorbance, molar absorptivity, and pathlength.
𝐴 =∈ 𝐵𝐶
2)
where A is absorbance, ∈ is molar absorptivity, B is pathlength, and C is concentration. Beer’s Law
was used to solve for concentrations using the absorbance data obtained at 540 nm and given a
specific path length of 1cm and a molar absorptivity of 1.03 x 104 L mol-1. These concentrations were
Concentration [M]
plotted against time to yield the spectra below in figures 3, 4, and 5.
0.00004
0.000035
0.00003
0.000025
0.00002
0.000015
0.00001
0.000005
0
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
Trial 6
0
45
89
134
179
Trial 7
Trial 8
Time (s)
Figure 3. Spectra of all solutions at 540 nm for red delicious apple
Concentration [M]
0.000025
Trial 1
Trial 2
0.00002
Trial 3
0.000015
Trial 4
Trial 5
0.00001
Trial 6
0.000005
Trial 7
Trial 8
0
0
42
84
126
168
Time (s)
Figure 4. Spectra of all solutions at 540 nm for golden delicious apple
Trial 9
Trial 10
0.00006
Trial 1
0.00005
Trial 2
Concentration [M]
Trial 3
0.00004
Trial 4
Trial 5
0.00003
Trial 6
0.00002
Trial 7
Trial 8
0.00001
Trial 9
0
0
37
74
111
Time (s)
148
185
Trial 10
Figure 5. Spectra of all solutions at 540 nm for granny smith apples.
The slope of these lines gives the rate of reaction for each concentration of each apple. Figure 6
below shows the dependence of rate on substrate concentration for each apple.
4.5E-08
4E-08
Rate mol L-1 S-1
3.5E-08
3E-08
2.5E-08
Granny Smith
2E-08
Red Delicious
1.5E-08
Golden Delicious
1E-08
5E-09
0
-5E-09 0
0.001
0.002
0.003
[Substrate] [M]
0.004
Figure 6. Comparison of rate to substrate concentration for each apple
Michaelis-Menten kinetics predicts that the rate of a reaction can be described using
equation 2 below
𝑉𝑜 =
𝑉𝑚 ∗[𝑆]
𝐾𝑚 +[𝑆]
2)
where Vo equals the rate found from the slopes of fig 4 and the similar graphs, Vm is the maximum
rate the reaction can achieve, Km is the substrate concentration at which the reaction rate is Vm/2,
and [S] is substrate concentration. This model yields figures 7, 8, and 9, which is the Mechaelis-
V0 [M/s]
Menten plot of each apple showing what Vo approaches at high concentrations.
0.000014
0.000012
0.00001
0.000008
0.000006
0.000004
0.000002
0
0
5
10
15
20
Substrate Concentration [M]
Figure 7. Golden Delicious Michaelis-Menten
0.000025
0.00002
V0 [M/s]
0.000015
0.00001
0.000005
0
0
5
10
15
20
Substrate Concentration [M]
Figure 8. Red Delicious Michaelis-Menten
V0 [M/s]
0.00002
0.000015
0.00001
0.000005
0
0
5
10
15
Substrate Concentration [M]
Figure 9. Granny Smith Michaelis-Menten
20
A Lineweaver-Burke plot plots 1/rate vs. 1/[S] for a variety of substrate concentrations with a
constant enzyme concentration. This is done by linearizing equation 2 into equation 3.
1
𝑉𝑜
=
𝐾𝑚
𝑉𝑚
1
∗ [𝑆] +
1
𝑉𝑚
3)
The intercept of this graph can be used to solve for Vm of each apple and the slope can then in turn
be used to determine the Km. The Lineweaver-Burke plot for each apple can be seen below in
figures 10 11 and 12.
250000000
1/V0 [s/M]
200000000
150000000
100000000
(1/V0) = 98681(1/[s]) + 2E+07
50000000
0
0
1000
2000
1/[S] (M-1)
3000
Figure 10. Lineweaver-Burke for golden delicious
1/V0 [s/M]
100000000
80000000
60000000
40000000
1/V0 = 25261(1/[s]) + 3E+07
20000000
0
0
1000
2000
3000
1/[S] (M-1)
Figure 11. Lineweaver-Burke for red delicious
80000000
1/V0 [s/M]
60000000
40000000
1/V0 = 26582(1/[s]) + 4E+07
20000000
0
0
500
1000
1/[S] (M-1)
Figure 12. Lineweaver-Burke for granny smith
1500
The data obtained from the Lineweaver-Burke plots can be seen more clearly in table 2
below. Notice also the slopes and intercepts in the table are more precise than the values on the
graphs because Microsoft Excel rounds the graphical values.
Table 2.
Calculated
Values
intercept
Granny
Smith
35990000
Red Delicious
25290000
Golden
Delicious
17890000
calc Vm (M/s)
slope
2.77E-08
26500
3.95E-08
25300
5.59E-08
98600
cal Km [S]
0.0007386
0.0009988
0.0055163
Since the maximum rate for granny smith is the lowest among the calculated values it follows that
the granny smith apple would brown the slowest and therefore would be the best apple for a fruit
salad.
Conclusion
The Vmax for granny smith was found to be 2.78 x 10-8 (M/s), the Vmax for red delicious was
found to be 3.95 x 10-8 (M/s). The Vmax for golden delicious was found to be 5.59 x 10-8 (M/s). The
Kmax for granny smith was 0.0007386 M, The Kmax for red delicious was 0.0009988 M, and the Kmax
for golden delicious was 0.0055163 M. From this data it can be seen that the granny smith is the
best for a fruit salad because both the Vmax and Kmax are lowest for granny smith.
References
1. Huisman, H. O. (1950), Investigations on quinones and quinone-derivatives ). Preparation and
antibiotic properties of some substituted p-benzo-and p-toluquinones, hydroquinones and
hydroquinone diesters. Recl. Trav. Chim. Pays-Bas, 69: 1133–1156.
doi: 10.1002/recl.19500690909
2. Laboratory Handout. “Which apple would be best for a Fruit Salad?” CHEM 403. Spring 2014.