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