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UMUC Biology 102/103
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you test to see if an enzyme was completely saturated during an
conditions that would alter the activity of an enzyme. Be specific with
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
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).
(1) 2 oz. Bottle (Empty)
(1) 100 mL Graduated Cylinder
30 mL Iodine-Potassium Iodide, IKI
2 Spray Lids
30 mL Starch (liquid)
*2 Food Products (e.g., ginger root, apple, potato,
Remove the cap from the starch solution. Attach
the spray lid to the starch solution.
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
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).
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.
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.
Select two food items from your kitchen cabinet
Obtain a kitchen knife and a cutting board. Carefully
cut your selected food items to create a fresh surface.
Figure 3:Sample set-up.
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).
Wash your hands with soap and water.
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.
Wait five minutes.
Hold the IKI spray bottle 25 – 30 cm away from
the paper towel, and mist with the IKI solution.
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.
Table 1: Substance vs. Starch Presence
Presence of Starch?
Positive Control: Starch
Negative Control:Student Must Select
What were your controls for
this experiment? What did they demonstrate? Why was saliva included in this
What is the function of
amylase? What does amylase do to starch?
Which of the foods that you
tested contained amylase? Which did not? What experimental evidence supports
Saliva does not contain
amylase until babies are two months old. How could this affect an infant’s
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?
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?
2: Effect of Temperature on Enzyme Activity
Yeast cells contain
catalase, an enzyme which helps convert hydrogen peroxide to water
Figure 4:Catalase catalyzes the decomposition of hydrogen peroxide
to water and oxygen.
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 +
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.
(2) 250 mL Beakers
30 mL 3% Hydrogen Peroxide, H2O2
20 cm String
3 Test Tubes (Glass)
Test Tube Rack
*You Must Provide
Use a permanent marker to label test tubes 1, 2,
and 3. Place them in the test tube rack.
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.
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.
Create a hot water bath by performing the
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).
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
If using a stove, place a small pot on the stove
and turn the stove on to a medium heat setting.
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.
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.
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.
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.
Let the tubes sit for 15 minutes.
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.
Carefully stretch the neck of the balloon to help
ensure it does not rip when stretched over the opening of the test tube.
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.
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.
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.
Swirl each tube to mix, and wait 30 seconds.
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.
Table 2: Balloon Circumference vs. Temperature
Balloon Circumference (Uninflated; cm)
Balloon Circumference (Final; cm)
1 – (Cold)
2 – (RT)
3 – (Hot)
What reaction is being
catalyzed in this experiment?
What is the enzyme in this
experiment? What is the substrate?
What is the independent
variable in this experiment? What is the dependent variable?
How does the temperature
affect enzyme function? Use evidence from your data to support your answer.
Draw a graph of balloon
diameter vs. temperature. What is the correlation?
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.
In general, how would an
increase in substrate alter enzyme activity? Draw a graph to illustrate this
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