Question;Your;Full Name;UMUC Biology 102/103;Lab4:Enzymes;INSTRUCTIONS;?;On your own and without;assistance, complete thisLab4AnswerSheet electronically and submit it via theAssignments Folder by the;date listedintheCourse;Schedule (underSyllabus).;?;To conduct your laboratory;exercises, use the Laboratory Manuallocated;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 Lab4AnswerSheet in the following format;LastName_Lab4 (e.g.;Smith_Lab4).;?;You should submit your documentas a Word (.doc;or.docx) or Rich Text Format (.rtf) file for best compatibility.;Pre-Lab;Questions;How could;you test to see if an enzyme was completely saturated during an;experiment?;List three;conditions that would alter the activity of an enzyme. Be specific with;your explanation.;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).;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;Negative Control:Student Must Select;Food Product;Food Product;Saliva;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;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 WaterBath;*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 ? 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);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?
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