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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?
Answer ) Saturation means that all of the enzyme molecules are filled with substrate and adding more substrate will not increase the rate of reaction.
2.List three conditions that would alter the activity of an enzyme. Be specific with your explanation.
Answer )
Temperature- Increase in temp causes increase in enzyme activity
pH- Most enzymes work at pH-7
Substrate concentration- An increase in substrate concentration causes an increase in enzyme activity because there more collisions with the enzymes and therefore faster reaction.
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.
Answer )
Detergents:
Enzymes have been used in many kinds of detergents to degrade proteins causing stains.
Foods and Beverages :
Enzymes are used to produce foods like wine, beer, vinegar and cheeses. Biotechnology has made it possible to isolate and characterize the specific enzymes responsible for these processes.
Textiles :
Enzymes are widely used to prepare the fabrics for clothing and to enhance the preparation of cotton for weaving by reducing impurities while improving the quality of the final textile product.
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).
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:
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 bottle.
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 or refrigerator.
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
Substance Resulting Color Presence of Starch?
Positive Control: Starch Dark Purple Yes
Negative Control : water yellow No
Food Product: Apple Yellow No
Food Product: Potato Dark Purple yes
Saliva: Amylase yellow 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?
Answer )The positive control is starch while the negative control is water. The control of an experiment is the part of an experiment that does not change and serve as reference points. Saliva is included in this experiment to demonstrate the action of salivary amylase.
2.What is the function of amylase? What does amylase do to starch?
Answer )Amylase digest the starch so that no purple / black color is produced in the presence of amylase. It would break down the starch molecules, and lugol’s solution would not change color.
3.Which of the foods that you tested contained amylase? Which did not? What experimental evidence supports your claim?
Apple contained amylase and was able to break down starch, so when sprayed with lugol’s solution the towel did not turn purple. Potato does not have amylase, the towel where it was rubbed turned purple after spraying it with lugol’s solution.
4.Saliva does not contain amylase until babies are two months old. How could this affect an infant’s digestive requirements?
Answer )
Since amylase is needed for digesting starches or complex carbohydrates, this would result in these types of foods not being digested and possibly passed right through the system.
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?
Answer )
Besides salivary amylase, which is for breaking down starches, the salivary gland produces salivary lipase, which is for breaking down fats into end products like fatty acids and diglycerides. It is produced by serous acini in the tongue and HCl in the stomach activates salivary lipase.
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?
Answer )
Pepsinogen is the inactive form of pepsin. The stomach has linings, which consist of various cells which secrete hydrochloric acid which when released inside the stomach activates pepsin to pepsinogen.
Thus, the activated proteases are in the lumen, or interior of the stomach, and are unable to digest the stomach. Both the stomach and small intestine have epithelial cells, which have tough membranes that provide resistance to these enzymes.
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.
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.
Escience Biology Lab Answer Key
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
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 following steps:
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 4e.
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
Tube Temperature (°C) Balloon Circumference (Uninflated; cm) Balloon Circumference (Final; cm)
1 – (Cold) 15 16.8
2 – (RT) 30 17.2
3 – (Hot) 80 6.9
Post-Lab Questions
1.What reaction is being catalyzed in this experiment?
Answer )2 H2O2 → 2 H2O + O2
2.What is the enzyme in this experiment? What is the substrate?
Answer )Catalase , hydrogen peroxide
3.What is the independent variable in this experiment? What is the dependent variable?
Answer )Temperature, enzyme activity
4.How does the temperature affect enzyme function? Use evidence from your data to support your answer.
Answer )Increasing the temperature will create the fastest reaction, because catalase are used to speed up biochemical reactions in the body. So they are used to working at body temperatures. However the cataylse do not react quicker than this at temperatures over 45ºC, because these temperatures will cause the catalase to denature, and therefore the reactions will be slower.
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.
Answer )
There is no negative control in this experiment. By introducing an enzyme Enterococcus faecalis which will show no reaction towards the substrate, we can revise this experiment to include the negative control.
7. In general, how would an increase in substrate alter enzyme activity? Draw a graph to illustrate this relationship.
Answer ) An increase in substrate concentration causes an increase in enzyme activity because there more collisions with the enzymes and therefore faster reaction.
7.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?
Answer)
Question: At what temperature is amylase, an important digestive enzyme in the human body, most active?
Enzyme: Amylase
Substrate: Starch
Hypothesis: Amylase has the fastest reaction rate at 15 C.
Independent variable: Temperature.
Dependent variable : reaction rate of the enzyme.
Controlled variables : Quantity of enzyme, the amount of iodine
Control: Amylase at room temperature
Procedure: This experiment was done five different times at five different temperatures to evaluate how temperature affects the reaction rate of amylase.
Instruments used:
Following equipments were available to help vary the temperature:
Ice (from the student freezer), the hot plate, and two water baths, one set at 37oC and the other set at 65oC. Room temperature is easy to achieve.
Escience Labs Manual Answers Biology Online
Procedure:
Put the thermometer directly in the amylase solution in the test tube.
When you achieve the desired temperature, leave the amylase solution at that temperature for at least 60 seconds, then remove the test tube.
Heat alone will make the starch break down above 50oC! If hot enzyme solution is added to starch and the color change is rapid and its hard to know whether the breakdown of starch is due to the elevated temperature or to the rate of the enzymatic reaction.
So to see if 65oC or 100oC denatures the enzyme, bring the temperature back to 37oC before combining it with the starch solution. The enzyme reaction at high temperature is not tested but whether the enzyme was denatured at that temperature is evaluated.
Thereafter, add the amylase solution to the starch + iodine, and time the reaction properly. Calculate the reaction rate. Assuming 2 mL of the 10 mg/mL starch solution was used, 20 mg starch in each reaction is calculated.
(2 mL x 10 mg/mL = 20 mg)
Reaction rate in mg/sec =
20 mg starch divided by the number of seconds until clearing
Lastly, the results are recorded in the data table, the reaction rate at each temperature is calculated and data graphed.