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BSC2010H Lab 4 - Enzyme Activity: Polyphenol oxidase

OBJECTIVE
Demonstrate knowledge of enzyme function and how function is affected by changes in
environmental conditions via an oral and written report evaluating experimental results.
INTRODUCTION
Thousands of chemical reactions are occurring in the cells in your body each
minute and all of them are controlled by biological catalysts called enzymes. Like all
catalysts, enzymes lower the activation energy of a reaction (i.e., the amount of energy
needed to trigger a reaction).
Enzymes are proteins, each with its own unique shape from its sequence of amino
acids. The shape of an enzymes active site determines its catalytic effects. The active
site of each enzyme will bind only with certain molecules and not others (Fig. 1).
Because of this, enzymes are said to be specific. A molecule that binds with an enzyme
and undergoes chemical modification is called the substrate of that enzyme.

Figure 1. Substrates enter the active site of enzymes where the


substrates are altered into products.
The binding between enzyme and substrate consists of weak, noncovalent
chemical bonds, forming an enzyme-substrate complex that exists for only a few
milliseconds. During this instant, the substrate is oriented so that it can be altered easily
by having bonds broken or made. The result is a chemical change in the substrate that
converts it to a new type of molecule called a product of the reaction. The product
leaves the enzymes active site, but the enzyme remains unchanged and can be used
again.
Individual enzyme molecules may enter the catalytic cycle several thousand times
per second; thus, a small amount of enzyme can convert large quantities of substrate to
product. Eventually enzymes wear out; they break apart and lose their catalytic capacity.
Cellular proteinases (more enzymes) degrade inactive enzymes to amino acids, which are
used to make other enzymes.
The amount of a particular enzyme found in a cell is determined by the balance
between the processes that degrade the enzyme and those that synthesize it. When no

enzyme is present, the chemical reaction catalyzed by the enzyme does not occur very
quickly. Conversely, if enzyme concentration increases, the rate of the reaction will also
increase until the amount of substrate becomes limiting.
Temperature and pH affect the ability of an enzyme to bind substrate by altering
the enzymes shape, including its active sites.
The Enzyme: Polyphenol oxidase (PPO)
Polyphenol oxidase (also called tyrosinase) is an enzyme found in many
biological materials. Normally it is a part of the pathway that catalyzes the reaction of the
amino acid tyrosine to form melanin a black pigment. The activity of the enzyme
contributes to the darkening of human skin in response to sunlight (increased production
of melanin), and the darkening (oxidation) of a freshly peeled potato when exposed to air.
The specificity of polyphenol oxidase for the substrate tyrosine is not great. It
will, in fact, recognize a variety of ring structures such as catechol in addition to its
natural substrate tyrosine.
Copy from the board the structures of tyrosine and catechol in the box below:

Tyrosine

Catechol

Orthoquinone

In your experiments today, you will use catechol which is converted to orthoquinone in
the presence of the enzyme. Orthoquinone immediately undergoes a second reaction
forming a colored product. Formation of the products occurs rapidly, and using a
spectrophotometer we can measure the change in color intensity over time and thus
obtain information on the rate of product formation. In other words, by calculating the
rate at which the color appears in the cuvette, we can ascertain the rate of enzyme
activity. Add the structure of product orthoquinone (also known as 1,2-benzoquinone)
to the box above.
In the space below name the substrate, enzyme, and product of the reaction we
will work with today:

------------------------->
substrate

enzyme

product

Materials A: Preparation of Crude Enzyme Extract


With the instructor's supervision, the entire class will perform steps 1 through 3 together
to prepare our crude enzyme extract. We will assign tasks to different students:
1) Carefully peel and rinse 2 medium sized potatoes. Cut the potatoes into small
pieces and place the pieces in a blender with 100 ml ice-cold distilled water (dH2O). Put
the lid on the blender and place an ice bag around the blender container. Using the
highest setting, homogenize the potato pieces for 30 - 60 sec. Filter the homogenate
through two layers of cheesecloth into an ice-cold 250 ml beaker. After filtration,
carefully pour about 45 ml of the filtered homogenate into each of two 50 ml chilled
centrifuge tubes. Keep the centrifuge tubes of homogenate on ice at all times.
2) Centrifuge the tubes in the 3rd floor Beckman L2 Centrifuge at 5000 x g for 5
min at 4C. Following centrifugation, use a pipette to remove the clear supernatant from
each tube (~ 10 to 20 ml), being careful to not disturb the pellet at the bottom of the tube.
Transfer the supernatant to a chilled 50 ml tube labeled "Supernatant." Keep this tube on
ice at all times.
3) Next, make 200 ml of a 5 % (v/v) solution of the supernatant with ice cold
distilled water (in the ice bucket on the instructors bench). Place this in a 250 ml
Erlenmeyer flask labeled "Enzyme." Keep this on ice too.
4) Bring 5 ml of "5% enzyme" to your bench in a 10 ml tube in an ice beaker.
* Why is it important to use ice-cold dH2O and to keep the homogenate, supernatant, and
crude enzyme on ice throughout the experiment?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
* What does filtering the homogenate through cheesecloth accomplish?
________________________________________________________________________
________________________________________________________________________
* What does centrifugation accomplish?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

Materials B: Preparation of the Buffered Substrate


Prepare a buffered substrate solution by adding to a 50 ml beaker:
5.0 ml 0.045 M catechol
5.0 ml 0.1 M sodium phosphate buffer pH 7.0 (already prepared on instructors bench)
15.0 ml dH2O (room temperature)
25.0 ml
The buffered substrate may be kept at room temperature during the entire lab.

* How many grams do I need to make 100 ml of 0.045M catechol if I know that the FW of
catechol is 110 g/mole?
________________________________________________________________________

* What is a buffer and how does it work?


________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
* Why is it important to have a buffer present in this experiment?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

Exercise I: The Basic Reaction


Your reaction mixture consists of:
1 ml of buffered substrate (catechol) + 1 ml of enzyme (PPO in potato extract)
At high concentrations of both substrate and enzyme, the amount of product formed (i.e.,
the change in absorbance) should be directly proportional to time. You will use the
spectrophotometers Kinetic Assay program to measure the absorbance at 540 nm once
every 30 seconds for 4 minutes. Using this wavelength will measure the change in
absorbance that results from formation of the colored product.

Procedure:
First, it is necessary to zero the instrument using a blank.
* What should you use as a blank? __________________________________________
Second, it is necessary to include a control along with the experimental reactions.
* What is a control?_______________________________________________________
* What controls should be used for this experiment?
Control 1_____________________________________
Control 2_____________________________________
(Hint: One control will test if any non-enzymatic oxidation of the substrate,
catechol, occurs. A second control is needed because the crude enzyme extract
contains both polyphenol oxidase and, inevitably, the natural substrate tyrosine.
This might result in the formation of a colored product.)
* What should you use as a blank for the controls? _________________________
1) Prepare 2 ml of your blank in the cuvette. Follow directions on the next page for setting
up a Kinetic Assay on your spectrophotometer.
2) Prepare your reaction mixture = 1 ml of enzyme + 1 ml of buffered substrate. Because
the enzyme must remain cold until the reaction starts, you should place the cuvette with 1
ml of buffered substrate into the spectrophotometer chamber, then add 1 ml of COLD
enzyme, pipeting up & down 1x to mix, right before pushing the <read sample> button.
3) Record the absorbance data over 240 seconds in Table I below.
4) Repeat with 2 more replicates of the reaction mixture, and then with the 2 controls.

Spectrophotometer Kinetic Assay:


<Kinetic> Yellow button
Kinetic Assay
Enter reading wavelength for kinetic assay: 540 nm
<Enter>
Enter duration of timed assay: 240 sec
<Enter>
Enter interval of data collection: 30 sec
<Enter>
Do you want to subtract background reading? No
<Enter>
Ready to read absorbance
Insert blank cuvette
<Read blank> Green button
Blanking Spec
A540 = 0.000
> = continue
Ready to read absorbance
Insert sample cuvette
<Read sample> Green button
When finished collecting absorbance data for 240 seconds, press <print> for report.

Table I. Absorbance of reaction at 540 nm over time


Time (seconds)
reaction
mixture

30

60

90

120

150

180

210

240

Change in
Absorbance
Abs Abs
4
1 min
min

rep 1
rep 2
rep 3
control
1
control
2
Average reaction rate = Absorbance/min _____________________________
5) Finally, calculate the rate of reaction for each reaction mixture (Abs 240 sec - Abs 0 sec =
Absorbance / 4 min), then convert to Abs/ min, and find the average for the 3 replicates.

6) Plot your data from all 3 replicates on graph paper or using Excel to illustrate the formation
of reaction products over time. Are the reaction rates during the first 30 seconds similar to the
last 30 seconds? What does this mean?

Exercise II: Reaction Rates Under Altered Conditions


Based on our discussions about proteins in general and enzymes in particular, you will next
test a set of reactions to observe enzyme activity under altered conditions. You will be
given a variable to test - a different pH, different temperature, or different enzyme
concentration.

Formulate a hypothesis which predicts whether the condition you change will
increase or decrease the polyphenol oxidase reaction rate observed in Exercise I.

State your hypothesis below before you begin your experiments:

___________________________________________________________________

Record your notes and data in your notebook as you carry out your study. You will
write up your results and conclusion to present orally as a powerpoint presentation
to the class next week, as well as a written lab report in 2 weeks.
Be prepared to compare your data from Exercise I: The Basic Reaction to the
data you collect in Exercise II: Reaction Rates Under Altered Conditions in your
presentation and lab report.

Table II. Absorbance of reaction at 540 nm over time


Time (seconds)
reaction
mixture

30

60

90

120

150

180

210

240

Change in
Absorbance
Abs Abs
4
1 min
min

rep 1
rep 2
rep 3
control
1
control
2
Average reaction rate = Absorbance/min _____________________________

* As in Exercise I, calculate the rate of reaction for each experimental (Abs 240 sec - Abs 0 sec
= Absorbance / 4 min), then convert to Abs/ min, and find the average for the 3 trials.

* Also plot your data from all 3 replicates on the same graph you made for Exercise I.

Are the
reaction rates during the first 30 seconds similar to the last 30 seconds? Are they similar to the

results from Exercise I? Discuss what these comparisons tell you in the conclusions of your
report.