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Pre-Lab Questions
1.
How could you
test to see if an enzyme was completely saturated during an experiment?
2.
List three
conditions that would alter the activity of an enzyme. Be specific with your
explanation.
3.
Take a look
around your house and identify household products that work by means of an
enzyme. Name the products, and indicate how you know they work with an enzyme.
Experiment
1: Enzymes in Food
This experiment tests for
the presence of amylase in food by using Iodine-Potassium Iodide, IKI. IKI is a
color indicator used to detect starch. This indicator turns dark purple or
black in color when in the presence of starch. Therefore, if the IKI solution
turns to a dark purple or black color during the experiment, one can determine
that amylase is not present (because presence of amylase would break down the
starch molecules, and the IKI would not change color).
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Materials (1) 2 oz. Bottle (Empty) (1) 100 mL Graduated Cylinder 30 mL Iodine-Potassium Iodide, IKI Permanent Marker Ruler 2 Spray Lids 30 mL Starch (liquid) *Cutting Board |
*2 Food Products (e.g., ginger root, apple, potato, etc.) *Kitchen Knife *Paper Towel *Saliva Sample *Tap Water *You Must Provide |
Procedure:
1.
Remove the
cap from the starch solution. Attach the spray lid to the starch solution.
2.
Rinse out the
empty two ounce bottle with tap water. Use the 100 mL graduated cylinder to
measure and pour 30 mL of IKI into the empty two ounce bottle. Attach the
remaining spray lid to the bottle.
3.
Set up a
positive control for this experiment by spraying a paper towel with the starch
solution. Allow the starch to dry for approximately one hour (this time
interval may vary by location).
4.
In the mean
time, set up a negative control for this experiment. Use your knowledge of the
scientific method and experimental controls to establish this component (hint:
what should happen when IKI solution contacts something that does not contain
starch?) Identify your negative control in Table 1.
Note: Be sure to space the positive and negative controls apart from
each other to prevent cross-contamination.
5.
When the
starch solution has dried, test your positive and negative controls. This step
establishes a baseline color scale for you to evaluate the starch concentration
of the food products you will test in Steps 7 – 11. Record your results in
Table 1.
6.
Select two
food items from your kitchen cabinet or refrigerator.
7.
Obtain a
kitchen knife and a cutting board.Carefullycut your selected food items to create
a fresh surface.
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Figure 3:Sample set-up. |
8.
Gently rub
the fresh/exposed area of the food items on the dry, starch-sprayed paper towel
back and forth 10 – 15 times. Label where each specimen was rubbed on the paper
towel with a permanent marker (Figure 3).
9.
Wash your
hands with soap and water.
10.
Take your
finger and place it on your tongue to transfer some saliva to your finger.
Then, rub your moistened finger saliva into the paper towel. Repeat this step
until you are able to adequately moisten the paper towel.
Note: You should always wash your hands before touching your tongue!
Alternatively, if you do not wish to put your hands in your mouth, you may also
provide a saliva sample by spitting in a separate bowl and rubbing the paper
towel in the saliva. Be sure not to spit on the paper towel directly as you may
unintentionally cross-contaminate your samples.
11.
Wait five
minutes.
12.
Hold the IKI
spray bottle 25 – 30 cm away from the paper towel, and mist with the IKI
solution.
13.
The reaction
will be complete after approximately 60 seconds. Observe where color develops,
and consider what these results indicate. Record your results in Table 1.
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Table |
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Substance |
Resulting |
Presence |
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Positive |
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Negative |
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Food |
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Food |
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Saliva: |
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Post-Lab Questions
1. What were your controls for this experiment? What did they
demonstrate? Why was saliva included in this experiment?
2. What is the function of amylase? What does amylase do to starch?
3. Which of the foods that you tested contained amylase? Which did
not? What experimental evidence supports your claim?
4. Saliva does not contain amylase until babies are two months old.
How could this affect an infant’s digestive requirements?
5. There is another digestive enzyme (other than salivary amylase)
that is secreted by the salivary glands. Research to determine what this enzyme
is called. What substrate does it act on? Where in the body does it become
activated, and why?
6. Digestive
enzymes in the gut include proteases, which digest proteins. Why don’t these
enzymes digest the stomach and small intestine, which are partially composed of
protein?
Experiment
2: Effect of Temperature on Enzyme Activity
Yeast cells contain
catalase, an enzyme which helps convert hydrogen peroxide to water
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Figure 4:Catalase catalyzes the |
and oxygen. This enzyme is
very significant as hydrogen peroxide can be toxic to cells if allowed to
accumulate. The effect of catalase can be seen when yeast is combined with
hydrogen peroxide (Catalase: 2 H2O2? 2 H2O + O2).
In this lab you will
examine the effects of temperature on enzyme (catalase) activity based on the
amount of oxygen produced. Note, be sure to remain observant for effervescence
when analyzing your results.
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Materials (2) 250 mL Beakers 3 Balloons 30 mL 3% Hydrogen Peroxide, H2O2 Measuring Spoon Permanent Marker Ruler 20 cm String |
3 Test Tubes (Glass) Test Tube Rack Thermometer Yeast Packet *Hot Water Bath *Stopwatch *You Must Provide |
Procedure
1.
Use a
permanent marker to label test tubes 1, 2, and 3. Place them in the test tube
rack.
2.
Fill each
tube with 10 mL hydrogen peroxide. Then, keep one of the test tubes in the test
tube rack, but transfer the two additional test tubes to two separate 250 mL
beakers.
3.
Find one of
the balloons, and the piece of string. Wrap the string around the uninflated
balloon and measure the length of the string with the ruler. Record the
measurement in Table 2.
4.
Create a hot
water bath by performing the following steps:
1.
Determine if
you will use a stovetop or microwave to heat the water. Use the 100 mL
graduated cylinder to measure and pour approximately 200 mL of water into a
small pot or microwave-safe bowl (you will have to measure this volume in two
separate allocations).
2.
If using a
stovetop, obtain a small pot and
proceed to Step 4c.If using a
microwave, obtain a microwave-safe bowl and proceed to Step 4e.
3.
If using a
stove, place a small pot on the stove and turn the stove on to a medium heat
setting.
4.
Carefully
monitor the water in the pot until it comes to a soft boil (approximately 100
°C). Use the thermometer provided in your lab kit to verify the water
temperature. Turn the stove off when the water begins to boil. Immediately
proceed to Step 5.
CAUTION:Be sure to turn the stove off after creating the hot water bath.
Monitor the heating water at all times, and never handle a hot pan without
appropriate pot holders.
5.
If using a
microwave, place the microwave-safe bowl in the microwave and heat the water in
30 second increments until the temperature of the water is approximately 100
°C. Use the thermometer provided in your lab kit to verify the water
temperature. Wait approximately one minute before proceeding to Step 5.
5.
Place Tube 1
in the refrigerator. Leave Tube 2 at room temperature, and place Tube 3 in the
hot water bath.
Important
Note:The water should be at approximately 85 °C when you place Tube 3
in it. Verify the temperature with the thermometer to ensure the water is not
too hot! Temperatures which exceed approximately 85 °C may denature the
hydrogen peroxide.
6.
Record the
temperatures of each condition in Table 2. Be sure to provide the thermometer
with sufficient time in between each environment to avoid obscuring the
temperature readings.
7.
Let the tubes
sit for 15 minutes.
8.
During the 15
minutes prepare the balloons with yeast by adding ¼ tsp. of yeast each balloon.
Make sure all the yeast gets settled to the bulb of the balloon and not caught
in the neck. Be sure not spill yeast while handling the balloons.
9.
Carefully
stretch the neck of the balloon to help ensure it does not rip when stretched
over the opening of the test tube.
10.
Attach the
neck of a balloon you prepared in step 8 to the top of Tube 2 (the room
temperature test tube) making sure to not let the yeast spill into the test
tube yet. Once the balloon is securely attached to the test tube lift the
balloon and allow the yeast to enter the test tube. Tap the bulb of the balloon
to ensure all the yeast falls into the tube.
11.
As quickly
and carefully as possible remove the Tube 1 (cold) from the refrigerator and
repeat steps 9 – 10 with Tube 1 using a balloon you prepared in step 8.
12.
As quickly
and carefully as possible remove Tube 3 (hot) from the hot water bath and
repeat steps 9 – 10 with Tube 3 using a balloon you prepared in step 8.
13.
Swirl each
tube to mix, and wait 30 seconds.
14.
Wrap the
string around the center of each balloon to measure the circumference. Measure the
length of string with a ruler. Record your measurements in Table 2.
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Table |
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Tube |
Temperature |
Balloon |
Balloon |
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1 – |
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2 – (RT) |
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3 – (Hot) |
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Post-Lab Questions
1. What reaction is being catalyzed in this experiment?
2. What is the enzyme in this experiment? What is the substrate?
3. What is the independent variable in this experiment? What is the
dependent variable?
4. How does the temperature affect enzyme function? Use evidence from
your data to support your answer.
5. Draw a graph of balloon diameter vs. temperature. What is the
correlation?
6. Is there a negative control in this experiment? If yes, identify
the control. If no, suggest how you could revise the experiment to include a
negative control.
7. In general, how would an increase in substrate alter enzyme
activity? Draw a graph to illustrate this relationship.
8. Design an experiment to determine the optimal temperature for
enzyme function, complete with controls. Where would you find the enzymes for
this experiment? What substrate would you use?
