Enzymes

UMUC Biology 102/103

Lab 4: Enzymes

INSTRUCTIONS:

· On your own and without assistance, complete this Lab 4 Answer Sheet electronically and submit it via the Assignments Folder by the date listed in the Course Schedule (under Syllabus).

· To conduct your laboratory exercises, use the Laboratory Manual located under Course Content. Read the introduction and the directions for each exercise/experiment carefully before completing the exercises/experiments and answering the questions.

· Save your Lab 4 Answer Sheet in the following format: LastName_Lab4 (e.g., Smith_Lab4).

· You should submit your document as a Word (.doc or .docx) or Rich Text Format (.rtf) file for best compatibility.

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).

concept_tab_2

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. Carefully cut your selected food items to create a fresh surface.

Figure 3: Sample set-up.
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.

Table 1: Substance vs. Starch Presence
Substance Resulting Color Presence of Starch?
Positive Control: Starch Dark Purple Yes
Negative Control : Cellulose Brownish red color No
Food Product: Apple Dark Purple yes
Food Product: Potato Dark Purple yes
Saliva: Amylase Brownish red color No

Post Negative Control -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

Figure 4: Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen.
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 + 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.

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:

a. 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).

b. 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.

c. If using a stove, place a small pot on the stove and turn the stove on to a medium heat setting.

d. 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.

e. 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.

Table 2: Balloon Circumference vs. Temperature
Tube Temperature (°C) Balloon Circumference (Uninflated; cm) Balloon Circumference (Final; cm)
1 – (Cold)
2 – (RT)
3 – (Hot)

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?