THE ACTIVITY OF LACTASE

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1 THE ACTIVITY OF LACTASE Lab VIS-8 From Juniata College Science in Motion Enzymes are protein molecules which act to catalyze the chemical reactions in living things. These chemical reactions make up the metabolism of the organism. There are two basic types of reactions that occur in living organisms: catabolic reactions and anabolic reactions. Catabolic reactions break larger molecules down into smaller molecules, anabolic reactions build larger molecules from smaller molecules. Lactase is a digestive enzyme found in the small intestine. This enzyme breaks down the sugar lactase into its two component sugars, glucose and galactose. Lactose is the principle sugar found in milk and is found in all dairy products. People who are lactose intolerant either lack lactase or have a defective version of the enzyme. These people cannot digest lactose and suffer various types of intestinal distress. People who are lactose intolerant commonly take a dietary supplement that contains lactase. Lactase is sold under the commercial names of Dairy Ease or Lactaid. Since milk is an opaque liquid, it is difficult to work with because there is no noticeable change. There is a substance called ONPG which can be used as a substitute for the milk. ONPG is a colorless liquid. Lactase splits ONPG into ONP and galactose. ONP is a clear yellow material, so we can observe a color change from clear to yellow and use a spectrophotometer to measure how much product is being produced. In this laboratory you will examine the effects of concentration, ph, temperature, and competing substrates on the activity of lactase. OBJECTIVES The student will be able to: 1. Define the terms enzyme and substrate. 2. Explain the result of increasing the enzyme concentration in a reaction. 3. Explain the result of increasing the substrate concentration in a reaction. 4. Discuss the effect of ph on the activity of lactase. 5. Discuss the effect of temperature on the activity of lactase. 6. Define the term competing substrate. 7. Explain the effect of a competing substrate on the activity of lactase. MATERIALS Lactase solution 0.5 Molar Lactose solution 5 millimolar ONPG solution Stop watch or clock Buffer solutions 2, 4, 6, 7, 8, 10, 12 Spectrophotometer 1M Sodium carbonate solution Test tubes 1 ml pipettes Distilled water 10 ml pipettes Cuvettes for spectrophotometer Test tube rack Graph paper Water baths 0 o C, 4 o C, 23 o C, 37 o C, 60 o C, 100 o C Westminster College SIM 1 of 9

2 PROCEDURE Tips for increased consistency of results: 1. Use a different pipet for each different solution. 2. While holding the test tube by the top, gently tap the bottom of the test tube. This will mix the solution, creating a uniform solution throughout the test tube. 3. Use caution while using the buffers in part D. Acids burn clothes and skin. A. Observing the reaction 1. Into a test tube place 4.5 ml of the ph 7 buffer solution and 0.5 ml of the ONPG. 2. Add 0.4 ml of the Lactase and observe the tube for several minutes. 3. Describe the appearance of the tube as the reaction occurs. PART 1 -- Effects of Concentration B. Effects of enzyme concentration 1. Turn on the spectrophotometer and set it to 420 nm. 2. Create a blank by adding 4.5 ml of ph 7 buffer, 0.5 ml of ONPG and 1 ml of the 1M sodium carbonate solution. 3. Fill one of the cuvettes about ¾ full with the blank solution. This is the blank cuvette. 4. Place the blank cuvette into the sample compartment of the spectrophotometer with the triangle on the cuvette facing the front of the instrument. Note: Before inserting a cuvette into the spectrophotometer, wipe it clean and dry with a kimwipe, and make sure that the solution is free of bubbles. Do not touch the clear sides of the cuvette. 5. Press 0 ABS 100%T. 6. Remove the blank cuvette from the instrument. 7. Set up eight clean test tubes and add 0.5 ml of ONPG to each, then add the following: Test Tube ph 7 buffer Test tube ph 7 buffer ml ml ml ml ml ml ml ml 8. Add the following amounts of lactase to each test tube and time the reaction for two minutes: Test tube Lactase Test tube Lactase ml ml ml ml ml ml ml ml Westminster College SIM 2 of 9

3 9. After timing the reaction for 2 minutes, add 1 ml of the 1M sodium carbonate solution to each test tube. This should stop the reaction. 10. Fill a cuvette with the solution from your reaction and place it into the spectrophotometer. Make sure that the triangle on the cuvette is facing the front of the instrument. 11. Record the absorbance for your solution in the Data Table for Part B. Note: The greater the absorbance, the greater the amount of product that was formed. 12. Collect the class data and draw a graph to show the effect of enzyme concentration on the reaction. C. The effect of substrate concentration 1. Use your blank from part B to rezero the absorbance of the spectrophotometer: a. Place the blank cuvette into the sample compartment of the spectrophotometer with the triangle on the cuvette facing the front of the instrument. b. Press 0 ABS 100%T. c. Remove the blank cuvette from the instrument. 2. Set up eight clean test tubes and add the following: Test tube ph 7 buffer ONPG Test tube ph 7 buffer ONPG ml 0.0 ml ml 2.0 ml ml 0.5 ml ml 2.5 ml ml 1.0 ml ml 3.0 ml ml 1.5 ml ml 3.5 ml 3. Add 0.4 ml of Lactase to each test tube and time the reaction for two minutes. 4. After timing the reaction for 2 minutes, add 1 ml of the 1M sodium carbonate solution to each test tube. This should stop the reaction. 5. Fill a cuvette with the solution from your reaction and place it into the spectrophotometer. Make sure that the triangle on the cuvette is facing the front of the instrument. 6. Record the absorbance for your solution in the Data Table for Part C. 7. Collect the class data and draw a graph to show the effect of substrate concentration on the reaction. PART 2 -- Effects of ph and temperature D. The effect of ph on enzyme activity 1. Create a new blank for the spectrophotometer by combining 4.5 ml ph 7 buffer, 0.5 ml ONPG and 1 ml 1M sodium carbonate. 2. Set the spectrophotometer to 420 nm. 3. Fill one of the cuvettes about ¾ full with the blank solution. This is the blank cuvette. 4. Place the blank cuvette into the sample compartment of the spectrophotometer with the triangle on the cuvette facing the front of the instrument. Note: Before inserting a cuvette into the spectrophotometer, wipe it clean and dry with a kimwipe, and make sure that the solution is free of bubbles. Do not touch the clear sides of the cuvette. 5. Press 0 ABS 100%T. 6. Remove the blank cuvette from the instrument. Westminster College SIM 3 of 9

4 7. Each group will set up six test tubes as follows: Test tube 4.5 ml 0.5 ml 1 ph 2 buffer ONPG 2 ph 4 buffer ONPG 3 ph 6 buffer ONPG 4 ph 8 buffer ONPG 5 ph 10 buffer ONPG 6 ph 12 buffer ONPG 8. Add 0.4 ml of Lactase to each of your test tubes and time the reaction for two minutes. 9. After timing the reaction for 2 minutes, add 1 ml of the 1M sodium carbonate solution to each test tube in order to stop the reaction. 10. Fill a cuvette about ¾ full with one of your solutions. 11. Place the cuvette into the spectrophotometer. Make sure that the triangle on the cuvette is facing the front of the instrument. 12. Record the absorbance for your solution in the Data Table for Part D. 13. Collect class data and draw a graph to show the effect of ph on the activity of the enzyme. E. The effect of temperature on enzyme activity 1. Use the blank from part D to rezero the spectrophotometer. a. Place the blank cuvette into the sample compartment of the spectrophotometer with the triangle on the cuvette facing the front of the instrument. b. Press 0 ABS 100%T. c. Remove the blank cuvette from the instrument. 2. Set up six test tubes by placing 4.5 ml of ph 7 buffer and 0.5 ml ONPG into each tube. 3. Place one of the test tubes into each of the following water baths: Test tube Water bath temp Test tube Water bath Temp 1 0 o C (ice bath) 4 37 o C 2 4 o C (ice water) 5 60 o C 3 23 o C (room temp) o C (boiling) 4. Allow the test tubes to remain at the temperature of the bath for at least 5 minutes. 5. While the test tubes are in the water baths, carefully add 0.4 ml of Lactase to each test tube and time the reaction for two minutes. 6. After timing the reaction for 2 minutes, add 1 ml of the 1M sodium carbonate solution to stop the reaction. 7. Fill a cuvette about ¾ full with the solution from your reaction. 8. Place the cuvette into the spectrophotometer. Make sure that the triangle on the cuvette is facing the front of the instrument. 9. Record the absorbance for your solution in the Data Table for Part E. 10. Collect class data and draw a graph to show the effect of temperature on the activity of the enzyme. Westminster College SIM 4 of 9

5 PART 3 -- Other factors (optional) F. Effect of a competitive substrate 1. Set up a new blank containing 4.5 ml ph 7 buffer, 0.5 ml ONPG and 1 ml 1M sodium carbonate solution. 2. Set the spectrophotometer to 420 nm. 3. Fill one of the cuvettes about ¾ full with the blank solution. This is the blank cuvette. 4. Place the blank cuvette into the sample compartment of the spectrophotometer with the triangle on the cuvette facing the front of the instrument. Note: Before inserting a cuvette into the spectrophotometer, wipe it clean and dry with a kimwipe, and make sure that the solution is free of bubbles. Do not touch the clear sides of the cuvette. 5. Press 0 ABS 100%T. 6. Remove the blank cuvette from the instrument. 7. Label two test tubes. 8. To test tube 1 add: 4.5 ml ph 7 buffer 0.5 ml ONPG 9. To test tube 2 add: 4.0 ml ph 7 buffer 0.5 ml ONPG 0.5 ml lactose solution 10. Add 0.4 ml of Lactase to each test tube and time for two minutes. 11. After timing the reaction for 2 minutes, add 1 ml of 1M sodium carbonate solution to each test tube in order to stop the reaction. 12. Fill a cuvette about ¾ full with the solution from test tube Place the cuvette into the spectrophotometer. Make sure that the triangle on the cuvette is facing the front of the instrument. 14. Record the absorbance for this solution in the Data Table for Part E. 15. Fill a second cuvette about ¾ full with the solution from test tube Place the cuvette into the spectrophotometer. Make sure that the triangle on the cuvette is facing the front of the instrument. 17. Record the absorbance for this solution in the Data Table for Part E. Westminster College SIM 5 of 9

6 Names Period Class Date THE ACTIVITY OF LACTASE RESULTS A. Observing the reaction -- Describe the change in the appearance of the reaction mixture as the reaction occurs. B. Effect of enzyme concentration C. Effects of substrate concentration Concentration Absorbance Concentration Absorbance 0.0 ml lactase 0 ml ONPG 0.5 ml lactase 0.5 ml ONPG 1.0 ml lactase 1.0 ml ONPG 1.5 ml lactase 1.5 ml ONPG 2.0 ml lactase 2.0 ml ONPG 2.5 ml lactase 2.5 ml ONPG 3.0 ml lactase 3.0 ml ONPG 3.5 ml lactase 3.5 ml ONPG Westminster College SIM 6 of 9

7 D. Effect of ph E. Effect of Temperature ph Absorbance Temperature Absorbance ph 2 0 o C ph 4 ph 6 ph 8 ph 10 ph 12 4 o C 23 o C 37 o C 60 o C 100 o C F. Competitive substrate Test Tube Substrate(s) Absorbance ml ONPG ml ONPG and 0.5 ml lactose Westminster College SIM 7 of 9

8 DISCUSSION 1. What happens to the reaction as you increase the amount of enzyme (Lactase)? 2. What happens to the reaction as you increase the amount of substrate (ONPG)? 3. At which ph(s) did the enzyme (Lactase) work the best? 4. At which ph(s) did the enzyme work the least? 5. How does ph affect enzymes? 6. At which temperature(s) did the enzyme work best? 7. At which temperature(s) did the enzyme work the least? 8. How does temperature affect enzymes? 9. What happened when you added lactose to the reaction mixture? 10. Why did the lactose affect the reaction? 11. Do you make Lactase? How do you know? Westminster College SIM 8 of 9

9 TEACHER INFORMATION Lab VIS-8 From Juniata College Science in Motion SAFETY NOTES This lab uses Ortho-NitroPhenol-beta-Galactopyranoside (ONPG) as a substitute for lactose. Since ONPG is a phenolic compound, it should be handled with caution. Do not dispose of the ONPG by dumping it into a sink drain. Instead, collect the used materials into a waste bottle and return it with your lab materials. The concentration of ONPG that we are working with is small (5 millimolar) and should not pose a problem to your students, however, if any gets onto the skin it is probably a good idea to make sure the student washes the affected area immediately. Any spills should be wiped up immediately with paper towels and the towels disposed of by placing them into the trash. Care should be taken when working with the ph buffer solutions and water baths in order to avoid skin burns. Students should wear protective goggles and possibly latex rubber gloves. TEACHING TIPS To save time and material, divide the class up into six lab groups. Each lab group will perform one of the variable tests. You can then collect class data and have the students analyze all the tests to explain the working of the enzyme. PREPARATION OF MATERIALS Lactase - Lactaid liquid, as you purchase it, will be too concentrated for use in this lab. Dilute the enzyme by placing 5 drops into 100 ml of distilled water. This dilution will provide an excellent reaction during the time period provided. ONPG - ortho-nitrophenol-beta-galactopyranoside - C 12 H 15 NO 8 -- To make a 5 millimolar solution dissolve 0.15 grams of ONPG in 100 ml of distilled water. Be sure to use fresh solution. The solution can be stored for about a month in a refrigerator and still remain good. Lactose - C 12 H 22 O To make a.5 Molar solution, dissolve 17.1 grams of lactose in 100 ml of distilled water. Stores indefinitely in the refrigerator. Sodium Carbonate - Na 2 CO 3-10H 2 O-- To make a 1 Molar solution, dissolve 28.6 grams of sodium carbonate in 100 ml of distilled water. Buffer solutions can be purchased from any scientific supply house or can be mixed on your own. Be sure to use clear buffers, not colored buffers. Colored buffers will affect the absorbance of the light in the spectrophotometer. Westminster College SIM 9 of 9

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