Intro Molecular & Cell Biology (Bio210A) Laboratory
Instructor: Elmar Schmid, Ph.D.
Lab Procedures: Catecholase Enzyme Experiments
Introduction: Catechol oxidase (CAT)

In the first part of our enzyme lab we will work with a member of a class of enzymes
called polyphenol oxidases (PPO)

Oxidases are enzymes which oxidize their substrate molecules by removing 1, 2 or
4 electrons which are transferred onto oxygen; they are oxidizing their substrate
molecules
- e.g. mitochondrial cytochrome c oxidase, NADPH oxidase, monoamino oxidase,
etc.

You will work with a PPO isolated from potato tuber which is called catechol
oxidase (or sometimes simply referred to as catecholase)

Catechol oxidase (CAT) is a copper (Cu2+)-containing enzyme, that catalyzes the
removal of 2 electrons and 2 hydrogens from catechol, a phenolic compound found
in many plant parts

In the presence of oxygen, the enzyme substrate catechol is converted into
benzoquinone, a brownish-colored molecule with a maximum absorption
wavelength λmax of 540nm, and water (see Graphic 1 below)

The brownish benzoquinone is responsible for the typical darkening of fruits or
vegetables after cutting or injury

Since benzoquinone is toxic to many herbivorous insects and shows microbicidal
(bacteria-killing) effects, scientists believe that the primary function of the catechol
oxidase reaction in plants is protection from bacterial and fungal pathogens as well
as insect attacks; it is their “natural antibiotic”!
In this part of the lab you will isolate catechol oxidase from potato tubers and use the
crude enzyme extract for experimental studies to answer following questions:
1. What is the Km of catechol oxidase regarding catechol? (= Procedure 1)
2. Do the molecules PTU, L-ascorbic acid and N-ethylmaleimide (NEM)
inhibit the catalytic activity of catechol oxidase? (= Procedure 2)
3. If yes, what kind of inhibition do the three compounds show (competitive,
non-competitive or allosteric)? (= Final Calculations & Result Analysis)
1
Intro Molecular & Cell Biology (Bio210A) Laboratory
Instructor: Elmar Schmid, Ph.D.
Graphic 1: The catalytic reaction of catechol oxidase
Catechol Oxidase
(PPO)
Cu2+
2 e-
O
OH
OH
+
O
½ O2
Catechol
Substrate
+
H2O
o-Benzoquinone
(brownish color, λmax = 540nm)
Product
• Catechol is a poison
• Avoid spills and contact with all catechol-containing solutions
• Do not mouth pipette catechol solutions
• Wear latex or vinyl gloves while working with catechol solutions
Graphic©E.Schmid/2003
Important data of the used chemicals and reagents in the lab:
- o-Benzoquinone stock solution: 1% (MW 108.1g)
- Catechol stock solution:
1% (MW 110.11g/mol)
- PTU stock solution:
1% (MW 152.2 g/mole)
- Molar extinction coefficient:
(o-benzoquinone; 540nm)
= _____ mM
= _____ mM
= _____ mM
εBQ = ________ cm-1 M-1
 you need these data for the final calculations and analysis in this lab
2
Intro Molecular & Cell Biology (Bio210A) Laboratory
Instructor: Elmar Schmid, Ph.D.
Procedure 1: Determination of the KM value of enzyme catecholase
 this part is done by all assigned lab groups!
1. Place 8 glass test tubes in a test tube rack and label them 1  8 using a marker
pen
2. Dilute the 1% catechol stock solution 1:10 in distilled water (dH2O) to prepare
your substrate working solution; you need 5ml
3. Pipette 5ml distilled water (dH2O) into each of the tubes, then pipette the
indicated volumes of catechol substrate working solution and catecholase
enzyme solution to the tubes following the pipetting scheme below
 make sure that you add your reagents from top to bottom (water first!)
Pipetting Table:
Tube #
1
2
3
4
5
6
dH2O [ml]
Catechol [μl]
Catecholase [μl]
4.5
500
-
4.9
100
4.85
50
100
4.8
100
100
4.65
250
100
4.4
500
100
Q.1: Which of your test tubes above serve as control(s)?
Q.2: Does your experimental design include a positive control?
7
8
3.9
2.4
1000 2500
100 100
__________________
____________
Q.3: If you answered no in Q.2, how could your positive control look like?
_____________________________________________________________________
4. Carefully mix the tube contents without spilling by either using a vortex or by
inverting head-over-head of the Parafilm covered tubes
 your instructor will show you how you do this!
5. Incubate the tubes for 10min at room temperature on your bench top then record
the absorbance in each of your tubes with the help of a spectrophotometer set at
a wavelength of 540nm
 if no spectrophotometer is available in the lab give each observed color change
in your test tubes a number between 1 and 10
 “0” = no color change (= blank)
 “10” = maximum observed color change in your series
6. Write your absorbance readings in the Result Table 1 below; then begin with the
second procedure as described below
 wait with your final calculations and analysis until you finished the second lab
activity with the catechol oxidase
3
Intro Molecular & Cell Biology (Bio210A) Laboratory
Instructor: Elmar Schmid, Ph.D.
Result Table 1:
Tube #
1
2
3
4
5
6
7
8
Absorbance (A540nm)
Color
(1  10)
Procedure 2: Inhibitor studies with the enzyme catecholase
 different groups will study different inhibitors; see tables below;
your instructor will tell which inhibitor you will be investigating
1. Place 8 glass test tubes in a test tube rack and label them 1  8 using a marker
pen
2. Pipette 5ml distilled water (dH2O) into each of the tubes, then add the enzyme,
the inhibitor and last the substrate catechol to the tubes following the pipetting
scheme below
 make sure that you add your reagents from top to bottom (water first!)
Inhibitor = PTU
Pipetting Table (Group A):
Tube #
1
2
3
4
5
6
7
8
dH2O [ml]
Catecholase [drops]
PTU [drops]
Catechol [drops]
5
10
5
10
10
5
10
20
1
5
10
20
2.5
5
10
20
5
5
10
20
10
5
10
20
25
5
10
20
50
Inhibitor = L-Ascorbic Acid
Pipetting Table (Group B):
Tube #
1
2
3
4
5
6
7
8
dH2O [ml]
Catecholase [drops]
L-Ascorbic Acid
5
-
5
10
-
5
10
20
5
10
20
5
10
20
5
10
20
5
10
20
5
10
20
10
10
1
2.5
5
10
25
50
[drops]
Catechol [drops]
Inhibitor = NEM
Pipetting Table (Group C):
Tube #
1
2
3
4
5
6
7
8
dH2O [ml]
Catecholase [drops]
NEM [drops]
Catechol [drops]
5
10
5
10
10
5
10
20
1
5
10
20
2.5
5
10
20
5
5
10
20
10
5
10
20
25
5
10
20
50
4
Intro Molecular & Cell Biology (Bio210A) Laboratory
Instructor: Elmar Schmid, Ph.D.
Q.1: Which of your test tubes above serve as control(s)?
__________________
Q.2: Does your experimental design include a positive control?
Which test tubes is it or are these? __________________________________
3. Carefully mix the tube contents without spilling by either using a vortex or by
inverting head-over-head of the Parafilm covered tubes
 your instructor will show you how you do this!
4. Incubate the tubes for 10min at room temperature on your bench top then
record the absorbance in each of your tubes with the help of a
spectrophotometer set at a wavelength of 540nm; pipette 1ml of your enzyme
reaction in the test tubes into a 1ml plastic or glass cuvet
 your instructor will show you how to operate the spectrophotometer and
how to do these important absorbance readings!
 if no spectrophotometer is available in the lab give each observed color change
in your test tubes a number between 1 and 10
 “0” = no color change (= blank)
 “10” = maximum observed color change in your series
5. Write your absorbance readings in the Result Table 2 below; then begin with
the calculations and data analysis as described below
 make sure you have a calculator ready
Result Table 2:
Tube #
1
2
3
4
5
6
7
8
Absorbance (A540nm)
Color
(1  10)
Final calculations & Result analysis
 this part is done again by all assigned lab groups!
1. Each group transfers its absorbance data for both experiments (= Part 1 and
2) into the Summary Data Sheet below; also write the amount of substrate (=
catechol) used in the different test tubes into the corresponding columns
2. Use the Lambert-Beer equation (see Introduction part or text below) to
calculate the concentration cBQ of benzoquinone (BQ) in each of your test
tubes; write the concentrations in μM into the Summary Data Sheet below
- Lambert-Beer equation:
A540 = εBQ x d x cBQ
 d = 1cm; εBQ =
cm-1 M-1
5
Intro Molecular & Cell Biology (Bio210A) Laboratory
Instructor: Elmar Schmid, Ph.D.
3. Calculate the catecholase enzyme activity ‘v’ for each tube in nmole / min
beginning with your known BQ concentration data for each tube
 remember: the total enzyme reaction time was 10 min!
 remember: your total reaction volume was 5ml
4. Build the reciprocal values for each, the enzyme activity v and the substrate
concentration CBQ and write it in the corresponding columns of the Summary
Data Sheet below
Summary Data Sheet:
Tube #
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Experiment 1
Substrate
Concentration (cCat)
Absorbance A540
cBQ [μM]
v [nmole/min]
1/v
1/cCat
Tube #
Experiment 2
Substrate
Concentration (cCat)
Absorbance A540
cBQ [μM]
v [nmole/min]
1/v
1/cCat
 use the data in the bold frame of the table to establish your Lineweaver-Burk plot!
5. Plot your calculated data in your Summary Data Sheet in form of two dot plots
showing :
a. v dependent on CBQ (+/- inhibitor)
= Graph A
b. 1/v dependent on 1/cBQ (+/- inhibitor)
= Graph B
= Lineweaver-Burk plot
 show your plots on a separate piece of paper
 hand-drawn or computer-build graphics are accepted!
6
Intro Molecular & Cell Biology (Bio210A) Laboratory
Instructor: Elmar Schmid, Ph.D.
6. Determine the Km (= Michaelis constant) and vmax (= maximum velocity) values
of the catecholase you used in your experiment
Km = ______ [
]; vmax = _______ [
]
7. Does catecholase enzyme have a high or low affinity for its substrate
catechol? Argue by comparing your enzyme Km with the Km’s of other known
enzymes!
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
8. Based on your Lineweaver-Burk plot findings and analysis, what kind of
catecholase inhibitor is the molecule you used in your enzyme inhibition
experiment?
Inhibitor molecule
(circle the one you worked with
below)
Ο
PTU
Ο
L-Ascorbic Acid
Ο
NEM
Type of catacholase inhibition
(circle the observed one below)
Ο
Ο
Ο
Ο
Ο
Ο
Ο
Ο
Ο
= competitive inhibitor
= non-competitive inhibitor
= Allosteric inhibitor
= competitive inhibitor
= non-competitive inhibitor
= Allosteric inhibitor
= competitive inhibitor
= non-competitive inhibitor
= Allosteric inhibitor
9. It is a common observation, that fruits and many tubers (e.g. potato!) of
plants are rich in L-ascorbic acid (vitamin C!!). Based on what you heard and
learned in this enzyme lab about the plant catecholase, could you come up
with a hypothesis about the possible role of ascorbic acid in fruits and
tubers? State a brief hypothesis below.
________________________________________________________________
________________________________________________________________
________________________________________________________________
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