Pre-Lab For Amylase Lab Goals: In this lab, students will study different aspects of enzyme activity by doing the following: A. Establish a method to measure the amount of maltose produced. B. Determine the effect of different amounts of enzyme on the rate of the reaction. C. Determine the effect of different amounts of substrate on the rate of the reaction. D. Determine the effect of pH and temperature on the rate of the reaction. Synopsis: This lab will help students learn about the nature of sugars and measurement of enzymatic activities affected by pH, temperature, and concentration of sugars. Students will also learn qualitative and quantitative techniques by using UV-Vis spectrophotometer, vortex mixers and pipet pump dispensers. Students will be able to plot and interpret data obtained from this laboratory exercise and come to conclusions about the activity of salivary amylase. Students' entering competencies: Before doing this lab, students should understand: o o o o o o o o
Safety The Nature of Carbohydrates Enzymes and Enzyme Activity Factors that affect the activity of enzymes: temperature, concentration, time Basic principles of spectrophotometry Liquid metric measurement and the correct tools for measurement: pipets, Brinkman pipet pump dispensers The use of vortex mixers and the importance of mixing soluions in this lab. Graphing
REAL WORLD APPLICATIONS 1. Industry: 2. Medicine 3. Water Quality 4. Food and Drug Administration)
Beer/Wine/food Bacteriology/Pharmaceutical To analyze food and drugs
SAFETY AND LAB TECHNIQUES Safety: 1. Avoid getting chemicals on hands. Wash hands immediately. 2. Do not drop glassware. 3. CAUTION: The water bath contains hot water. Steam will burn. 4. Be careful when using test tubes in hot water bath. Use a test tube holder. Lab Techniques: 1. Label all test tubes carefully. Be sure you can identify your set of test tubes. (Class period, group name or number, test tube number: A1, A2, A3, A4, etc.) 2. Avoid contamination. 3. Hold cuvette on the ribbed part to avoid getting fingerprints on clear surface. 4. Use Kimwipes only to clean fingerprints off clear surface of cuvette. 5. When vortexing, hold test tube at the tip and tilt it away from you. 6. Be careful to add all solutions to test tubes. Check off as you go along. 7. Use one cuvette only when using the spectrophotometer. 8. Be sure to fill the cuvette at least 3/4 full. 9. After using spectrophotometer, pour liquid back into the original test tube. 10. Rinse cuvettes with deionized water before reusing. 11. Before reusing the cuvette, tap excess water out of it. Do not use paper towels, kimwipes, or any other material to wipe cuvette. 12. When placing cuvette in the spectrophotometer, make sure the ribbed side faces the front. 13. Remember timing is critical. 14. Emphasize to students the need to cool test tubes to room temperature. Suggested Pre-lab Demonstrations and Related Activities or the teacher 1. Demonstrate technique of using Spectrophotometer with a Spec-20. 2. Use a flashlight or projector to shine light through a liquid with different concentrations of food coloring to demonstrate absorption. 3. Test a cracker with no salt (or Matzoh). Chew and hold for about two
minutes. Check for starch conversion to sugar by taste. 4. Sugar + Benedict's solution in test tube = no color change Matzoh + Benedict's in test tube + boiling = no color change Chewed matzoh + Benedict's + boiling = color change to orange 5. Extra credit: students bring in labels/containers of various foods with sugars. 6. Demonstration: Cooked liver + H2O2 = little reaction, little O2 Raw liver + H2O2 = more reaction, more O2 7. Plotting a graph activity.
FACTORS AFFECTING THE ACTIVITY OF SALIVARY AMYLASE PRELAB HANDOUT: WORKSHEET
1. a) There are many macromolecules whose names end in the suffix "-ose." These organic molecules are called: ____________________ b) There are many macromolecules whose names end in the suffix "-ase." These organic molecules are called: ____________________ 2. Name three factors that affect the rate of enzyme activity. a)___________________________________ b)___________________________________ c)___________________________________ 3. a) The water bath in this lab procedure is set for what temperature? ____________(units) b) This temperature is:
cool to the touch
4. Convert the following: a) 1000 µL= ______________mL b) _____________ µL= 20 mL c) _____________ µL= 1.0 mL 5. Practice graphing the following data on to a blank graph. Amount of fructose (µL) Absorbance 0 0.00009 100 0.18247 300 0.20962 500 0.23454 700 0.29006 1000 0.41083
Place the absorbance on the Y axis and the amount of fructose on the X axis. Draw a smooth curve through the points. Be sure to include your units. 6. Tubes #A1, B1, and C1 contain no maltose, enzyme, or substrate. The purpose of these tubes is to serve as the ________________________ 7. When placing the cuvette into the spectrophotometer for analysis: a) the sample must fill at least ____________ of the cuvette; b) the light must pass through the ____________ side of the cuvette. 8. There is one chemical in this lab that especially deserves caution because it is a skin irritant. The name of this chemical is: ________________________
9. Use your textbook to look up examples of the following:
a) monosaccharide b) disaccharide c) polysaccharide Enzymes a) oral cavity (mouth) b) stomach 10. Fill in the following table upon completion of the spectrophotometer analysis. Each group will place their data on the board when they are done.
TUBE # A1 A2 A3 A4 A5 B1 B2 B3 B4 B5 C1 C2 C3 C4 C5 D1 D2 D3
Spectrophotometer Reading (Absorbance)
Factor That Was Varied For This Group
Comments on Results
Pre-Lab Quiz: Enzyme Action 1. In the reaction: H-R-OH + H2O2 → R-O + 2 H2O if the enzyme peroxidase is involved: a. What are the substrates?
b. What are the products?
2. The colorless molecule guaiacol reacts with hydrogen peroxide (H2O2) to form a colored compound. What is the purpose of this reaction in our experiment and how is the spectrophotometer involved?
3. What do you think will happen to the rate of reaction in question #1 if the amount of enzyme is increased?
4. What do you think will happen to the rate of reaction in question #1 if the amount of substrate is increased?
5. What do you think will happen to the rate of reaction in question #1 if the temperature of the reaction is increased to l00oC?
POST-LAB CRITICAL THINKING ACTIVITY - AMYLASE LAB Student directions: You have just finished a lab activity in which you experimented with various factors that affect the activity of salivary amylase. Evaluate the following experiment and see if you can draw a conclusion based on the background and data provided. This will provide you with an opportunity to see if you can apply the principles learned in the lab to a new situation. How do digestive enzymes function in Paramecia? Paramecia ingest food particles and enclose them within food vacuoles. Each food vacuole circulates in the cell as the food is digested by enzymes that are added to the vacuole. Nutrients made available during digestion are absorbed into the cytoplasm. Analysis 1. Some digestive enzymes function best at higher pH levels, while others function best at lower (more acid) pH levels. 2. Congo red is a pH indicator dye; it is red when the pH is above 5 and blue when the pH is below 3 (very acid). 3. Yeast cells that contain Congo red can be produced by adding dye to solution in which the cells are growing. 4. When paramecia feed on dyed yeast cells, the yeast is visible inside food vacuoles. 5. Examine the drawing below. The appearance of a yeast-filled food vacuole "over time" is indicated by the colored circles (labeled due to black and white drawing) inside the paramecium. Each arrow indicates movement and that time has passed.
SEE TOPS WEB SITE
Analyze what happens to the pH in the food vacuole over time. Explain your conclusions about the sequence of different digestive enzymes that function in paramecium digestion.
POST-LAB CRITICAL THINKING ACTIVITY Student directions: You have just finished a lab activity in which you experimented with various factors that affect the activity of salivary amylase. Evaluate the following experiment and see if you can draw a conclusion based on the background and data provided. This will provide you with an opportunity to see if you can apply the principles learned in the lab to a new situation.
How does dilution affect enzyme action? Starch digestion begins in the mouth with enzymes that are secreted in saliva. You decide to see what effect diluting the saliva will have on the digestion of soda crackers, which are mostly baked flour.
Analysis To verify that crackers contain starch, you grind up a cracker and add 5 mL of plain water. You add a drop of iodine solution, and it produces a dark blue color, indicating the presence of starch. Next, you repeat the test but use 5 mL of saliva instead of water. This time, the iodine produces a grayish-purple color instead of the blue color of a positive starch. Apparently, your saliva enzymes had broken down all the starch in the time it took to make the mixture and test it. Now you decide to see what effect dilution of saliva will have on the digestion of the starch. You place one drop of saliva into 5 mL of water in a test tube, and add the same amount of ground cracker as before. You test the tube at various time intervals and obtain the data shown in the following table:
Iodine Test for Starch
Critical thinking Draw a conclusion about the effect of dilution of the enzyme found in saliva on the digestion of starch, and explain the results.
Amylase Lab Post Lab Activity 1. What is an enzyme? What is the function of an enzyme?
2. Describe the role of each of the following compounds in the Amylase lab: A. DNS reagent B. Amylopectin C. Amylase D. Maltose E. Distilled Water
3. Restate the purpose of the lab; then design a flowchart which represents the difference between parts A, B, C, and D.
4. Looking at the lab, do test tubes A1, Bl, and Cl have anything in common? If so, what? Why is this similarity necessary in the lab?
5. What would the contents of a test tube be if it had an absorbance of 0.00?
6. Looking at the flowchart below, what would happen if you eliminated step two of part B or part C?
Add DI H2O, amylopectin, and
→ minutes → and titrate → Vortex → Water → Spectrophotometer
amylase to test tubes C1-C5
7. Why did you use the hot water bath?
8. What would happen if you left the lid off the hot water bath during the five minute time period?
9. The relationship between substrates, enzymes, and products can be represented by the following equation:
enzyme substrate → product
In a complete sentence, write this equation using the substrate, enzyme, and product in the amylase lab.
10. If the optimum temperature for human chemical activity is body temperature (36.6°C), what would you hypothesize would happen to the rate of reaction of amylase as it approached 0°C? What would you hypothesize would happen as the temperature approached 40°C? Explain.
Use Graph A to answer questions 11-15. 11. What is the substrate? What is the product?
12. Does the amount of substrate depend upon the amount of product, or does the amount of product depend upon the amount of substrate? Explain your answer.
13. What is the relationship between the amylopectin and the maltose?
14. Where did the information on the volume of maltose come from?
15. According to Graph A, what would happen if you continued to increase the volume of amylopectin? Why? What does this have to do with "maximum rate"?
Use Graph B to answer questions 16-18. 16. Which line is more reasonable? Why?
17. What is the relationship between Line A and volume of maltose?
18. What does line B indicate?
EQUIPMENT SKILLS CHECKLIST. Have your teacher initial next to each piece of equipment as you demonstrate its proper use.
____________Brinkman pump ____________cuvette ____________loading UV-Vis spectrophotometer & scanning sample
LACTASE ENZYME LAB Lactase is an enzyme that works on lactose, a sugar. Lactose sugar is a disaccharide, two rings big. To digest it, you need to break it down into two pieces, two monosaccharides. Lactase enzyme allows you to do this. It reduces the amount of energy needed for the hydrolysis of the disaccharide into two monosaccharides. Enzymes are proteins. Temperature can effect them. If you cook egg white protein, it changes from clear and runny to firm and white. It is denatured. Enzymes must fit with their substrate in order to work. If an enzyme is denatured, it cannot fit anymore. An enzyme that cannot fit, cannot do its job. We will test the effect of temperature on how well lactase enzyme works. First, we must have a way to test whether the enzyme is working or not. We will use an indicator that changes color. TesTape is the indicator. It changes color in the presence of glucose, a monosaccharide. IF THE ENZYME IS WORKING, IT WILL PRODUCE GLUCOSE. IF THE ENZYME IS NOT WORKING, IT WILL NOT PRODUCE GLUCOSE.
We will see how well the enzyme works after being subjected to different treatments. We will test: 1. milk + enzyme (our control) 2. milk + boiled enzyme (Does boiling stop the enzyme from working?) 3. boiled milk + enzyme (Does boiling the substrate stop the enzyme from working? Hint: is the substrate a protein or a sugar?) 4. plain, untreated milk (Does milk have glucose without enzyme treatment?)
All the milk we are testing has had enzyme added to it already, because the enzyme works too slowly for us to add it in class. To test the various combinations, make a serial dilution of the enzyme-treated milk: pure, 1:10, 1:100. Do this for each of the 4 kinds of combinations. Test each with TesTape. Repeat for 3 trials of each combination.
SAFETY: TesTape IS FOR EXTERNAL USE ONLY. IT DOES NOT BELONG NEAR YOUR MOUTH. WASH HANDS WITH SOAP AFTER YOU ARE DONE. TesTape is used by people who need to test the amount of sugar in their urine because they have diabetes. If they find sugar, it means they have too much in their blood and they must do something about it, such as take insulin. Thus, TesTape is a very sensitive test for glucose. Lives depend on it working well. Most people make lactase enzyme when they are young. As they get older, milk is not as important a part of their diet. Many people no longer make lactase as they get older. They then have trouble digesting dairy products. The lactase enzyme they can no longer produce for themselves can be added to dairy products before they eat them. That is why it was easy to buy lactase for the experiment. Do not try adding lactase to food without following package directions and without parental supervision.
LACTASE LAB EXTENSION THE EFFECT OF pH ON LACTASE ACTION Extend your lactase lab to investigate the effects of pH on lactase action. Most enzymes can only do their work within a relatively narrow range of pH values. Should the environment become too acidic or too alkaline (too basic), the enzyme becomes denatured and cannot work. Different enzymes work best under different conditions. One that must work in the acidic environment of the stomach differs in this way from one that operates in the more basic interior of the small intestine. Using a pH meter or universal indicator paper, make a vinegar/distilled water solution of pH 3. Make a sodium bicarbonate /distilled water solution of pH 10. Test a control solution. In the bottom right well of your culture plate, put 5 drops of distilled water and 1 drop of lactose solution. Test the pH of the mixture. Add one drop of lactase solution. Wait 5 minutes and test for glucose production with TesTape. Starting from well A-l, make a serial dilution of the vinegar solution from pure to 1:1000. Add a drop of lactose solution to each well. Test and record the pH of each well. Add a
drop of lactase solution to each well. After 5 minutes, test each mixture for glucose production with TesTape. Repeat the vinegar test for 2 more rows. Did you get similar results each time? Make a serial dilution of the sodium bicarbonate/distilled water solution from pure to 1:1000. Add a drop of lactose solution to each well. Test and record the pH of each well. Add a drop of lactase solution to each well. After 5 minutes, test each mixture for glucose production with TesTape. Repeat the sodium bicarbonate solution for 2 more rows. How could you modify this experiment to make it more precise? more accurate? What other acids and bases could make the experiment more realistic? Could any other chemical interactions modify your results in the changed experimental conditions?
AMYLASE ENZYME LAB VOCABULARY BUILDERS BG VF TA NE AQ TZ CW AA EC RI ND TK LP RP XQ
ABSORPTION ACID AMYLASE AMYLOPECTIN BASE CATALYST
V P R M M C N K T D E M O R N
W X T Y F P N C U A H F E O F
X V R Z J W E S O T L A M D K
I Z A N V P B R O W I Y I U E
T D T E O N L G A O S W S C L
OZ R RYM E YN LZP OCI I GO VT S AAB RP N T OH L UJ US R LP I TJ O ZRW
DEIONIZED DIGESTION DNS ENZYME MALTOSE PHOTO
A S L L B N H H R P W L E C I
G X W U D M O C B O L U L I A
U M F X Y T D I G E S T I O N
S P H F D R M O E S A B D R R
O A M Y L A S E L D H N A G E
PRODUCT REACTANT SUGAR TARTRATE TEMPERATURE ULTRAVIOLET