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Looking Into Lactase An Enzyme Lab Involving Medical Biotechnology

Maryland Loaner Lab Teacher Packet

Adapted by Jennie Queen-Baker for the Maryland Loaner Lab Program at UMBI

Table of Contents T E A C H E R M A T E R I A L S

Loaner Lab Overview 1 Maryland Science Content Standards 2 Introduction 3 Pre-laboratory Activity 4 Laboratory Explanation 7 Answers to Analysis Questions 9 Teacher Laboratory Preparation 11 Extension Activities 18 Post-Laboratory Activity: Bioscience… Real Jobs, Real People 23

S T U D E N T A C T I V I T Y H A N D O U T S A N D L A B O R A T O R Y P R O T O C O L

Memo 5 Used in Pre-Laboratory Activity Medical Fact Sheet 6 Used in Pre-Laboratory Activity Laboratory Protocol 12 Used in the Laboratory Activity

Developed from the University of Maryland Biotechnology Institute's "Program to Enhance the Understanding of Biotechnology for Baltimore City High School Students" funded through a grant from the Eisenhower Professional Development Program. The original lab module was called “Medical Biotechnology: Using Living Things as Tools to Solve Medical Problems”.

MdBio, Inc. (www.mdbio.org) has awarded a $50,000 grant to University of Maryland Biotechnology Institute’s Maryland Loaner Lab Program so schools that do not have access to MdBioLab can still perform Looking Into Lactase for free with their students.

The MdBioLab is sponsored by: Fisher Scientific, Inc. NIH Foundation

SAIC Bio-Rad Montgomery County, MD

Looking Into Lactase Page 1

Looking Into Lactase Loaner Lab Overview

The Looking Into Lactase lab has two parts: • A Pre-Laboratory activity that allows students to explore the condition of lactose intolerance and

becoming familiar with the symptoms associated with it. • A laboratory activity that allows students to work with an enzyme and to investigate the changes in

physical conditions that may alter its activity. Also, students make a connection between laboratory science and an application for medical biotechnology.

Teachers and students who will be performing the Looking Into Lactase lab activity using the Maryland Loaner Lab must first complete the Pre-Laboratory classroom activity. The conceptual aspects of the curriculum will be reinforced with the laboratory activity. Maryland Loaner Lab Supplies: Description Quantity Comments Must be Returned Plastic capped tubes labeled “#1,#2, and #3” (empty)

30 For experiment #1—for milk samples #1,#2, and #3

Yes

Glucose test strip bags 10 One for each station Yes Glucose test strips (in bags) 150 15 strips in each bag Yes, if unused. Pipet pumps 10 One for each station Yes Plastic 10 ml pipettes 20 10 “Lactex”, 10 “Cow milk” Yes Plastic 10 ml pipettes (labeled “Cow, Rice, Soy”)

3 Used by teacher to fill tubes #1,#2, and #3 with milk samples

Yes

“Lactex” 15 ml conical tubes 10 Filled with 15 ml of lactase, Keep Refrigerated until use.

Yes

Plastic capped tubes filled with Range of pH solutions

60 10 sets of pH solutions: 2,4,7,10,12, and dH2O. Keep at room temperature.

Yes

Plastic capped tubes labeled “RTM,RTL,CM,CL,HM,HL” (empty)

60 For experiment #3—for milk and lactex solutions at room temperature, cold, and hot temps.

Yes

“Cow Milk” 50 ml conical tubes (empty)

10 To be filled by the teacher with 20 ml of cow milk

Yes

White plastic tube floater 1 For experiment #3, to float tubes in hot water bath

Yes

Thermometer 1 For experiment #3 Yes

Teacher Supplies: Description Quantity Comments Paper towels 10 One for each station Tube racks 10 One for each station Rice milk 25 ml Used in experiment #1 Cow milk 450 ml Used in experiment #1,#2, and #3 Soy milk 25 ml Used in experiment #1 Goggles Enough for entire

class Used in experiment #2

Ice in containers (or use of freezer in the lab)

Enough to share with 10 stations

Used in experiment #3

Water bath or hot plate with 1000 ml glass beaker

1 Used in experiment #3 (see bottom page 11)

Looking Into Lactase Page 2

Looking Into Lactase Maryland Science Content Standards These classroom and laboratory activities meet several of the Maryland Science Content Standards: Goal I: Skills and Processes The student will demonstrate ways of thinking and acting inherent in the practice of science. The student will use the language and instruments of science to collect, organize, interpret, calculate, and communicate information. Expectation 1.2 The student will pose scientific questions and suggest investigative approaches to provide answers to questions. Expectation 1.3 The student will carry out scientific investigations effectively and employ the instruments, systems of measurement, and materials of science appropriately. Expectation 1.4 The student will demonstrate that data analysis is a vital aspect of the process of scientific inquiry and communication. Expectation 1.5 The student will use appropriate methods for communicating in writing and orally the processes and results of scientific investigation. Expectation 1.7 The student will show that connections exist both within the various fields of science and among science and other disciplines including mathematics, social studies, language arts, fine arts, and technology. Goal 3: Concepts of Biology The student will demonstrate the ability to use scientific skills and processes and major biological concepts to explain the uniqueness and interdependence of living organisms, their interactions with the environment, and the continuation of life on earth. Expectation 3.1 The student will be able to explain the correlation between the structure and function of biologically important molecules and their relationship to cell processes. Goal 4: Concepts of Chemistry The student will demonstrate the ability to use scientific skills and processes to explain the composition and interactions of matter in the world in which we live. Expectation 4.1 The student will explain how the observations of the properties of matter forms the basis for understanding its structure and changes in its structure. Expectation 4.5 The student will investigate the impact of Chemistry on society.

Looking Into Lactase Introduction

The Looking Into Lactase laboratory is an activity to facilitate learning about substances called enzymes (their activities and things that affect them) and the connection of laboratory science and the study of medical biotechnology. This laboratory contains an activity that teaches the student how to use the lactase enzyme to break down lactose in milk samples. It also investigates the effects of pH and temperature on enzyme activity. And finally, there is a connection throughout the laboratory to the medical condition of lactose intolerance with an explanation and understanding of its symptoms. The pre-laboratory activities include a storyline, which involves “Pharmex Pharmaceuticals” which is a fictitious company. As the teacher, you are to act as the Principal Investigator working in this company overseeing the various research teams (students). You are to tell the students that this company is currently working on the development of a tablet to be used by people who suffer symptoms of lactose intolerance. The tablet would contain lactase. In the scenario, the name of the tablet is given as Lactex®, but in reality the commercially available Lactaid Ultra® tablet is used in the investigations. What are Enzymes? Enzymes are organic catalysts that control the rate of chemical reactions in cells while not being permanently altered themselves. In general, enzymes speed up the rate of reaction by lowering the activation energy required to start reactions. Enzymes are extremely efficient. They can catalyze reactions at rates up to 10 billion times higher than comparable non-catalyzed reactions. By their specific configuration, they hold the reactant molecules in close proximity and in the correct orientation for the reaction to occur. Each enzyme has a specific site (the “active site”) where the substrate and enzyme combines. Enzymes also show specificity in that each specific type of enzyme acts on a particular substrate or on a certain kind of chemical bond. The specificity of enzymes is due to their structures. Each enzyme has a characteristic three-dimensional (3-D) shape. The analogy of a “lock and key” is often used to illustrate the temporary joining of a specific enzyme to a specific substrate in an enzyme-substrate complex. When an enzyme is subjected to high temperatures, the enzyme ceases to function (There are exceptions such as the enzymes in thermophilic bacteria). This is due to the denaturing of the enzyme (unfolding of the 3-D structure). At lower temperatures, the 3-D structure is intact, but there may not be enough energy for a reaction to occur. In this case the enzyme is inactive, but not permanently denatured. What is Lactase? Lactase is an enzyme that is used by the body to hydrolyze lactose, a disaccharide unique to mammalian milk, into the monosaccharides glucose and galactose. Lactose has been shown to aid in the absorption of several minerals, including calcium, magnesium, and zinc.

Introduction Page 3

Looking Into Lactase Pre-Laboratory Activity NOTE: Groups using the Maryland Loaner Lab must first complete the pre-laboratory classroom activities. The conceptual aspects of the curriculum will be reinforced with the laboratory investigation.

Engage: (30-60 minutes) Introduce the scenario of the students working for a pharmaceutical company and read out loud the Memo from the Principal Investigator (Teacher) (page 6). Develop further the scenario of a pharmaceutical company involved in the development of a new product (a tablet containing the lactase enzyme) that can be used by people who experience the symptoms of lactose intolerance. This lab is an example of Medical Biotechnology. 1. Hand out a copy of the Medical Fact Sheet (page 7). Have the students read the sheet and engage

them in a short discussion (personal experiences, people they know, what they might have already learned about the topic, etc.) about lactose intolerance.

2. Discuss and/or review information about enzymes including their structure, function, and how they are

affected by various conditions. Have the students gain a deeper understanding of enzyme structure and function by using other materials which would include the following information about enzymes: a. Enzymes are organic catalysts, speeding up chemical reactions while not being permanently

altered themselves. b. The specificity of enzymes is due to their structures. Each enzyme has a characteristic three-

dimensional (3-D) shape. c. The analogy of a “lock and key” can be used to illustrate the temporary joining of an enzyme to a

specific substrate. d. When an enzyme is subjected to high temperatures, the enzyme ceases to function. This is due to

the unfolding of the 3-D structure and the enzyme is said to be denatured. At lower temperatures, the 3-D structure is intact, but there may not be enough energy for a reaction to occur. In this case the enzyme is inactive, but not permanently denatured.

3. Have students read the first paragraph from each of the three experiments to introduce the scenarios to

the students. Discuss the goals of the three experiments, and have the students make predictions (hypotheses) for each.

Other suggested activities for the students: 1. Have students write an entry in a laboratory notebook explaining the purpose of the three experiments

to be performed in this laboratory exercise. 2. Have students interpret a graph. 3. Have students construct a graph. 4. Have students compare human digestion with that of cows and sheep. 5. Have students relate lactose tolerance to evolution.

Pre-Laboratory Activity Page 4

Pharmex Pharmaceuticals MEMO

To: All Research Team Members From: Principal Investigator Date: 10/10/05 Re: Testing the efficacy of our new enzyme product

I commend all of you research team members for your hard work to date. I thought it might help to remind you why it is so important for us to work diligently for the successful development of our Lactex® product. I have enclosed a flier that I picked up at a doctor’s office, which includes a Medical Fact Sheet. Please share this flier with your team members. Our newest product, Lactex®, is nearly ready for the last phase of testing before submission to the U.S. Food and Drug Administration for approval. Once we get the FDA’s approval, we can begin large scale manufacturing and marketing of our product. Before this, however, we need to verify Lactex®’s ability to break down the disaccharide lactose into glucose and galactose. We will also need to check Lactex®’s ability to function at different temperatures and different pH values. You are to work in your teams to conduct three experiments. Please submit your final laboratory protocols, which include your results, after completion. Also remember to follow established safety procedures, as we do not want any fines assessed us by OSHA (Occupational Safety and Health Administration).


Medical Fact Sheet What is Lactose Intolerance? Lactose intolerance is a medical condition that is due to little or no activity of the enzyme lactase, which prevents lactose from being broken down into glucose and galactose. With low lactase activity in the small intestine, undigested lactose is passed into the colon where bacteria ferment the sugar to hydrogen gas and organic acids. Gastrointestinal symptoms of lactose intolerance can include bloating, abdominal pain, cramps, gas, flatulence, and sometimes diarrhea. The symptoms usually are noticed anywhere from 30 minutes to 2 hours after the ingestion of dairy products containing lactose. The severity of symptoms varies by individuals depending upon how much lactose is consumed and whether it is consumed alone or with other foods; and on the type of dairy products, because some dairy products contain more or less lactose. For example certain hard or aged cheeses like cheddar and Swiss contain less lactose than drinking a glass of milk, and therefore are easier to tolerate. Yogurt, which contains active bacterial cultures, is better tolerated as well because the yogurt contains enzymes that digest lactose. What Diseases May be Caused by Lactose Intolerance? Avoiding dairy products may result in an inadequate dietary intake of calcium, which may lead to various diseases including rickets, osteoporosis, and hypertension. Dairy foods are an excellent source of natural calcium, protein, vitamins A and D and other nutrients. Calcium is especially important for healthy bones and teeth. Milk is the primary source of calcium for children. It is necessary that children who are lactose-intolerant obtain the necessary calcium through some supplemental means. What Medical Care is Available? Forms of medical care for those suffering from the symptoms of lactose intolerance include the manipulation of the diet by carefully eliminating certain milk-containing products. Another form of medical care is the use of supplemental lactase derived from yeast and other fungal species. These can be in the form of liquid drops or chewable tablets. Individuals can take these prior to the ingestion of lactose-containing foods. There are also lactase supplemented milk and dairy products now available. What Causes Lactose Intolerance? The gene for the production of lactase is located on chromosome two. However there does not seem to be any difference in the DNA of individuals with or without lactase activity. Rather the differences are in the messenger RNA (mRNA), which may indicate that the primary regulation of this enzyme occurs during translation. Who Is Lactose Intolerant? Both childhood- and adulthood-onset of lactase deficiency are very common and are inherited as autosomal recessives. Those individuals who have lactase activity that persists into adulthood have inherited at least one dominant gene. Patterns of inheritance show racial variability. Northern Europeans have the lowest prevalence of adult-onset lactase deficiency at approximately 5%. Central Europeans have a prevalence of about 30%. Southern Europeans, as well as Hispanic and Jewish populations, show a high prevalence of approximately 70%. While Northern Indians show a25% prevalence, Southern Indians show a much higher prevalence of 65%. Both Asians and Africans are nearly all (90%) lactose intolerant as adults.


Looking Into Lactase Laboratory Explanation The purpose of the laboratory activity is to apply the concepts developed in the Pre-Laboratory to an investigation about the function of enzymes and the connection of laboratory science (research and development of products) to medical biotechnology. The objectives of the laboratory activity are as follows:

• Students will determine the effect of the lactase enzyme on 3 types of milk by conducting an experiment (students will work in groups).

• Students will determine the effect of pH and temperature on the activity of the lactase enzyme by conducting two additional experiments.

Before proceeding with the laboratory investigation, it is necessary to make a logical connection to the concepts developed in the Pre-Laboratory. In doing so, the laboratory activity becomes a tool in the continuum of an ongoing problem rather than an isolated end in itself. Developing the Concept for the Laboratory Activity Facilitate a discussion of how to differentiate the various types of milk listed in experiment #1, because the milk samples look very similar. Steer the discussion towards analyzing the different types of milk based on the different carbohydrates contained in each, specifically looking for cow milk that contains lactose. After establishing the use of lactase on the milk samples, discuss as a class how changing the physical environment (such as pH and temperature) may affect the activity of lactase as questioned in experiments #2 and #3. Reasons for checking pH and temperature in this scenario would be to make sure the Lactex® tablet could be taken with acidic foods or basic things such as lemons or antacids, or could be taken with hot or cold foods such as hot soup or ice cream. Make sure the students have an understanding of what pH is. Discuss what neutral, acidic, and basic (or alkaline) pH values are and where they are found in the pH range (0-14).

The Laboratory Investigation All three experiments will use glucose test strips to detect the presence of glucose in the samples. An enzymatic reaction takes place on the test pad of the glucose strip, causing the yellow test pad to turn a shade of green if glucose is present. The shade of green corresponds to the relative amount of glucose detected (up to 3000 mg/dl), and can be compared to the color chart provided on the bag. It is important to dip the test pad completely into the samples, or else an incomplete reaction may occur and inaccurate results obtained. Strips that are dipped into a sample should not touch another strip, or contamination of samples may happen. Experiment #1 “Identify the Cow Milk”, has students testing unknown milk samples (rice, cow, and soy milk) before and after the addition of lactase, to determine the identity of each. They will be provided with a chart that lists the type of sugar contained in each type of milk, and with the test results they should be able to identify the three unknown milk samples. Experiment #2 “Determine Effect of pH on Lactase Activity”, will involve students testing a range of different pH solutions mixed with cow milk, to see if there is a change in the lactase activity. Experiment #3 “Determine Effect of Temperature on Lactase Activity”, has students testing cow milk and lactase samples that have been incubated at room temperature, hot temperatures, and ice cold temperatures to determine if the lactase activity is affected.

Laboratory Explanation Page 7

Equipment Note: How to Use a Pipette Pump Pipette pumps will be used with plastic 10 ml pipettes to measure liquids. Secure the plastic pipette into the pump by using a pushing and twisting motion. Use the wheel to draw liquid into the pipette by rolling it forward, reverse the wheel’s direction to let the liquid out. Always hold the pipettes when attached to the pump upright, or the liquid will go inside the pump and become contaminated and volume will be lost. The 10 ml plastic pipettes have two scales on them that run in opposite directions. When measuring liquid, use the scale that has the “1 ml” at the bottom tip and “10 ml” at the top. Also make sure to use the bottom of the meniscus (the curved part of the liquid in the pipette) to determine the volume level. When transferring liquid, make sure the container you are transferring the liquid into is nearby or liquid may start to drip out of the pipette tip. The transfer must take place quickly.

The labeled pipettes should be used only with the corresponding liquids (they can be reused with the same liquid only), otherwise the pipettes and samples risk contamination. When using the pipettes the tip shouldn’t touch the insides of tubes you are dispensing the liquid into, to also prevent contamination. Interpretation of Results In experiment #1, the only sample that should contain glucose and the strip turn green (very dark in color, ~3000 mg/dl) before the lactase is added, should be tube #1 that contains rice milk. Rice milk contains only glucose and that is why the strip turns green before adding lactase. After adding lactase both tubes #1 and #2 will have detected glucose and the strips turned green (again #1 is very green and #2 will be a medium green, ~300 mg/dl). The actual amounts of glucose detected may vary slightly, as the time incubation may be slightly different between student groups. Tube #2 that contains cow milk turns the test strip green after the addition of lactase because it contains lactose. Tube #3 when tested before and after adding lactase, never turns a test strip green and stays yellow. Tube #3 has soy milk that contains sucrose. In experiment #2, the color results for the glucose test strips should be medium green (~300 mg/dl) for pH solutions 2, 4, and 7. Solutions above a pH of 7 did not detect glucose and the strips remained yellow. The optimum range for the activity of lactase would be considered pH 2-7. The water control tube should match the tube with a pH solution of 7, which should be a medium green color on the test strip. In experiment #3, the results may vary due to fluctuation in temperatures. If the cold tubes are kept in the freezer at -20°C (or on ice) and the hot tubes kept in a water bath at 70-75°C both for 10 minutes, then the best results will be obtained. The lactase enzyme works best at room temperature and should detect glucose on the test strip at a medium green color (~300 mg/dl). At very cold temperatures the lactase works, just not as well. The energy required by the enzyme to function is not as available when the temperatures are cold. The test strip at cold temps should detect glucose but at a lesser amount (~100 mg/dl). At very hot temperatures, the lactase enzyme did not work at all. The enzyme is denatured at very hot temperatures and inactivates it. The test strip at hot temps did not detect glucose and remained yellow in color. Results for both experiments #2 and #3 could be graphed. The laboratory protocol asks the students to identify both the dependent and independent variables for each of the three experiments. This should aid the students in preparing a graph, so they know on which axis to plot their data.

Laboratory Explanation Page 8

Looking Into Lactase Answers to Analysis Questions (From Student Worksheets)

Part 1: Identify the Cow Milk Identify the independent and dependent variable: The independent variable is the set of conditions that is manipulated, in this case the unknown milk samples. The dependent variable is the set of conditions that is measured, in this case the lactase activity (as shown by the presence of glucose). 1. What is the purpose of the glucose test strips?

The strips serve as an indicator for the presence of glucose, which is one of the products of lactase activity. 2. What effect does the lactase have on the cow milk?

The lactase catalyzed the hydrolysis of the disaccharide, lactose, giving its two monosaccharides, glucose and galactose. 3. Which is the cow milk? How do you know?

Sample #2. There was no measurable glucose in the original sample but after adding the lactase there was measurable glucose. This was the only solution that had these results. (Sample #1 was rice milk and # 3was soy milk)

4. Can Lactex® be used specifically on lactose? Are there any effects on the other sugars? Yes, because there was no change in the amount of glucose measured on the rice and soy samples. The lactase did not have any effects on the sugars in rice or soy milk.

Part 2: Determine Effect of pH on Lactase Activity Identify the independent and dependent variable: The independent variable is the pH and the dependent variable is the lactase activity (as shown by the presence of glucose). When graphing the results, the independent variable is on the X axis and the dependent variable is on the Y axis. 1. Which solution represents the control(s)?

The water sample represents the control (the pH is 7). 2. What is the optimum pH range for Lactex®? How do you know?

The optimum pH for the enzyme appears to be pH 2 through pH 7 because there were glucose readings of 300mg/dl in that range and above pH 8 there was no glucose.

3. What effect does changing the environment from an alkaline (basic) environment to an acidic environment have on the activity of the lactase enzyme? The lactase enzyme worked well in the acidic environment (pH<7) but did not work at all in the alkaline environment (pH>7). So going from an alkaline environment to an acidic one, makes the enzyme active.

4. Do you think it is possible to bring the pH back to the optimum range and restore enzyme activity? Since the structure of the enzyme is not changed by the pH, it is possible to adjust the pH back to the optimum range and restore enzyme activity.

Part 3: Determine Effect of Temperature on Lactase Activity Identify the independent and dependent variable: The independent variable is the temperature and the dependent variable is lactase activity (as shown by the presence of glucose). When graphing the results, the independent variable is on the X axis and the dependent variable is on the Y axis. 1. Which temperature represents the control?

Room temperature represents the control. Experiments #1and 2 were performed at room temperature.

Answers to Analysis Questions Page 9

2. What is the optimum temperature for Lactex®? How do you know? The enzyme appears to work best at room temperature. The highest glucose reading occurred at room temperature (300mg/dl).

3. Explain the difference in enzyme activity at varying temperatures. What happened to the enzyme? At room temperature the enzyme worked very well. At a colder temperature the enzyme worked but not as well as it did at room temperature. At the highest temperature the enzyme did not work at all. At the higher temperature the enzyme no longer broke down the lactose into glucose and galactose. At a cold temperature, there was not enough energy for the enzyme to work. At the higher temperature, the enzyme denatured which destroyed the 3-D structure making it incapable of interacting with lactose.

4. Do you think it possible to recover the enzyme activity from the ones treated to extreme temperature conditions? Why or why not? It is possible to recover the enzyme activity from the cold temperature since the structure didn’t change. By adding energy (heat), the enzyme should work well, again. The enzyme exposed to extreme heat is not recoverable since it was denatured. Cooling it down does not allow the enzyme to reform the proper 3-D structure to make it functional.

Answers to Analysis Questions Page 10

Looking Into Lactase Teacher Laboratory Preparation Prepare Student Stations (for 10 groups): Paper towel Pipette pump 2 plastic 10 ml pipettes labeled “Lactex” and “Cow milk” Brown bags with 15 glucose test strips (color chart on bags) Conical tube with 15 ml “Lactex” solution Goggles (especially for experiment #2) Test tube rack (with 3 rows each) for capped test tubes:

Front row: (exp. #1) Unknown milk samples (filled by teacher, see below) in tubes labeled “#1,#2, and #3” Middle row: (exp. #2) pH solutions and water control in filled tubes labeled “2,4,7,10,12, and dH2O” Last row: (exp. #3) Milk and “Lactex” at various temps in tubes labeled “RTM, RTL, CM, CL, HM, HL”

Conical tube with 20 ml “Cow Milk” (Hand out when starting exp. #2). (filled by the teacher, see below) Student copies of the laboratory protocol Shared equipment for multiple groups: Thermometer Ice in buckets/containers (or use of freezer) Water bath or hot plate with a 1000 ml glass beaker (white plastic tube holder used in either one) Step 1 – Preparing unknown milk samples (Tubes #1,#2, and #3) Using 3 labeled plastic pipettes “Cow, Soy, and Rice”, add 2 ml of each of the appropriate unknown milk samples (should be at room temperature) to their corresponding test tubes (labeled #1, #2, and #3). Sample #1 is rice milk, sample #2 is cow milk, and sample #3 is soy milk. Step 2 – Filling “Cow Milk” conical tubes Using the same labeled plastic pipette from Step 1 for cow milk, fill the conical tubes labeled “Cow Milk” with 20 ml each. Helpful Hints: Experiment #1 and #2 need to have reagents at room temperature. Allow milk samples, lactase solution, and pH solutions to warm to room temperature before starting the lab. WARNING: Experiment #2 uses acids and bases for the pH solutions. Goggles must be worn at all times during this experiment. Instruct students to notify a teacher if the pH solutions are spilled, and to wash hands immediately. Experiment #3 works best if students identify their tubes with a group number, they will be placing their tubes in ice buckets and a hot water bath. Assign group numbers when starting Experiment #3. It is possible to not use ice and place tubes in a rack that will be kept in a freezer, if nearby. A water bath with an adjustable heater or a hot plate with a 1000 ml glass beaker with hot water can be used for the hot temperature section. The water bath being used for the hot temps should be at 70-75°C and not above 75°C or tubes will melt and warp (use thermometer provided to monitor). Tubes should be heated without caps. All hot tubes should be placed in the provided tube holder so that they are floating in the water bath, not resting on the bottom of a heated container or they will also melt and warp (if using a glass beaker, it must be 1000 ml). The teacher should monitor the hot water bath at all times keeping student safety in mind.

Activity: Time needed:Preparing samples & student stations

30 minutes

Pre-Lab activity 30 minutes Laboratory activity 90 minutes Post-Lab activity (video) 10 minutes

Teacher Laboratory Preparation Page 11

Looking Into Lactase Laboratory Protocol

Part 1: Identify the Cow Milk

I

I D

T12

3

4

5

T1

2

Laborat

1. Identify the independent and dependent variables:

You are a research scientist at a company called Pharmex. We need to verify the activity of lactase found in our new tablet called Lactex®. The tablet must be tested on different types of milk that contain different sugars, to verify that it only works on lactose. If the enzyme has additional activities that affect other sugars, it may not be approved for use by the FDA. Three milk samples will be tested: cow, soy, and rice. This is a blind test to avoid bias, so the samples are labeled #1, #2, and #3. It is your job to determine which sample contains the different types of milk and to prove that Lactex® breaks down only lactose.

Milk Sugar Cow LactoseSoy SucroseRice Glucose

dentify the independent and dependent variables:

ndependent Variable: __________________________

ependent Variable: ___________________________

Lactose

Lactase

galactoseglucose

est unknown milk samples for glucose . Each station has 3 unknown milk samples labeled #1, #2, and #3. . Label three glucose test strips individually as # 1, # 2, and #3. Do not touch the test pad with your

hands. Close container after you remove strips. . Take glucose test strip #1 and dip it into tube #1 (just enough to get the test pad wet). Immediately

remove the test strip and lay it down on the paper towel. Repeat for samples #2 and #3. Keep the strips separated on the paper towels.

. Wait 3 minutes and record the color of the test strip in Table 1. This gives a base-line amount of glucose before anything has been done to the milk samples.

. Compare the color that develops on the test strip to the color chart on the test strip bag and record the relative amount of glucose in Table 1. Set aside the used glucose strips, start a “throw away” pile.

est unknown samples for lactase enzyme activity . Identify the tube labeled “Lactex®” and mix gently by inversion to get an even distribution of the

powder through the solution. Do not shake vigorously or produce bubbles as this may damage the enzyme.

. Using the pipette labeled “Lactex®” add 1 ml of the solution to tubes #1, #2, and #3. Remember DO NOT TOUCH tubes with the end of the pipette or your samples will be contaminated (use proper pipette technique).

ory Protocol Page 12

3. Cap tubes #1, #2, #3 and invert to mix a few times but do not shake vigorously. 4. Wait 3 minutes. 5. Remove 3 new glucose test strips. Label all the strips individually as #1, #2, and #3. 6. Take new glucose test strip #1 and dip it into tube #1 (just enough to get the test pad wet). Immediately

remove the test strip and lay it down on the paper towel. Repeat for samples #2 and #3. Keep the strips separated on the paper towels.

7. Wait 3 minutes and record the color of the test strip in Table 1. This shows any breakdown of the sugar to produce glucose.

8. Compare the color that develops on the test strip to the color chart on the test strip bag and record the relative amount of glucose in Table 1.

9. Identify the milk contained in samples #1-3 in Table 1 below by using the test results and the chart provided in the introduction.

6. Set aside the used glucose strips, add to the “throw away” pile.

Table 1: Glucose Results Before and After Adding Lactex®

Sample Number

Color of test strip BEFORE adding lactase, AFTER dipping in milk samples

Relative amount of glucose (mg/dl) BEFORE adding lactase

Color of test strip AFTER adding lactase, AFTER dipping in milk samples

Relative amount of glucose (mg/dl) AFTER adding lactase

Milk Type

1

2

3

Analysis Questions – Part 1

1. What is the purpose of the glucose test strips?

____________________________________________________________________________

____________________________________________________________________________

2. What effect does Lactex® have on cow milk?

____________________________________________________________________________

____________________________________________________________________________

3. Which sample is the cow milk? How do you know?

____________________________________________________________________________

____________________________________________________________________________

4. Can Lactex® be used specifically on lactose? Are there any effects on the other sugars?

____________________________________________________________________________

____________________________________________________________________________

Laboratory Protocol Page 13

Part 2: Determine Effect of pH on Lactase Activity

We are now certain our Lactex® tablet breaks down only lactose. You have been asked as the research scientists, to determine under what conditions it will work. Since the tablet is ingested, it must be determined whether or not the Lactex® tablet can remain effective through a wide range of pH values that it may encounter with different foods and liquids while entering the human digestion tract. Using cow milk, your team will conduct an experiment to test the activity of the Lactex® tablet across a range of pH values.

pH=0 pH=7 pH=14

Acidic Neutral Basic

Identify the independent and dependent variables: Independent Variable: __________________________ Dependent Variable: ___________________________ Add lactase to the appropriate tubes 1. Each station has 5 tubes containing 1 ml of pH solutions “2”, “4”, “7”, “10”, and “12” and one tube

containing “dH2O” which is distilled water. 2. Identify the tube labeled “Lactex®” and again mix gently by inversion to get an even distribution of the

powder through the solution. 3. Using the pipette labeled “Lactex®,” add 1 ml of the lactase solution to all 6 tubes. DO NOT

TOUCH the end of the pipette inside the tubes or your samples will be contaminated (use proper pipette technique).

4. Cap all the tubes and invert to mix a few times but do not shake vigorously. 5. Wait 5 minutes.

Add cow milk to tubes 1. Using the pipette labeled “cow milk,” add 2 ml of cow milk to all 6 tubes. DO NOT TOUCH the end

of the pipette inside the tubes or your samples will be contaminated (use proper pipette technique). 2. Cap all the tubes and invert to mix a few times but do not shake vigorously. 3. Wait 3 minutes.

Test for glucose 1. Label six new glucose test strips with the labels found on the tubes: “2,4,7,10,12,and dH2O”. 2. Take glucose test strip labeled “2” and dip it into the tube with pH solution “2” (get the test pad wet).

Immediately remove the test strip and lay it down on the paper towel. Repeat for the other samples. Keep the strips separated on the paper towels.

3. Wait 3 minutes and record color of the test strip and corresponding glucose amount in Table 2. 4. Set aside the used glucose strips, add to the “throw away” pile. 5. After completing the experiment, as a post-laboratory activity you will generate a line graph that shows

the dependence of enzyme activity on pH. Be sure to put a title on the graph and label the axes.


Table 2: pH and Glucose Results pH Color of strip Relative amount of glucose

(0-3000mg/dl) after adding lactase 2

4

7

10

12

dH2O control

Analysis Questions – Part 2

1. Why do we use the water sample?

_______________________________________________________________________

2. What is the optimum pH range for Lactex®? How do you know?

_______________________________________________________________________

_______________________________________________________________________

3. What effect does changing the environment from an alkaline (basic) environment to an acidic environment have on the activity of the lactase enzyme? ________________________________________________________________________

________________________________________________________________________

4. Do you think it is possible to bring the pH back to the optimum range and restore enzyme

activity?

________________________________________________________________________

________________________________________________________________________


Part 3: Determine Effect of Temperature on Lactase Activity

As research scientists at Pharmex, we are certain the Lactex® tablet breaks down lactose and have determined which pH range the tablet works best. You have been asked to determine under what temperature conditions the Lactex® tablet will work. The tablet may sometimes be ingested with cold foods (such as ice cream) and then go into the stomach (a generally warm place). It must be determined whether or not the Lactex® tablet can remain active in a variety of temperatures. Using cow milk, your research team will test the activity of the Lactex® tablet at different temperatures. The temperatures being tested in the lab are: freezing cold (ice), very hot (boiling water), and room temperature.

Identify the independent and dependent variables:

Independent Variable: __________________________ Dependent Variable: ___________________________

Set up the tubes for temperature incubation 1. Each station has six test tubes labeled: “HL” (hot Lactex®), “HM” (hot milk), “RTL” (room temp

Lactex® ), “RTM” (room temp milk), “CL” (cold Lactex®), and “CM” (cold milk). Put your group number on all tubes.

2. Identify the tube labeled “Lactex®” and again mix gently by inversion to get an even distribution of the powder through the solution.

3. Using the pipette labeled “Lactex®”, add 1 ml of lactase solution to the three tubes labeled: HL, RTL, and CL

4. Using the pipette labeled “cow milk”, add 2 ml of cow milk to tubes labeled: HM, RTM, CM. Place the test tubes at their respective temperatures for 10 minutes: (NO CAPS)

a. hot tubes HL and HM into the hot water b. cold tubes CL and CM into the ice c. room temperature tubes RTL and RTM leave in the rack at your station.

5. Record the hot water temperature, the ice temperature, and room temperature in °C in Table 3. 6. Carefully pour the contents of the HM tube into the HL tube. DO NOT SPILL. Tubes and contents will

be hot so use appropriate laboratory safety techniques, hold tubes at the top. 7. Carefully pour the contents of the CM tube into the CL tube. DO NOT SPILL. 8. Carefully pour the contents of the RTM tube into the RTL tube. DO NOT SPILL. 9. Cap all the tubes and invert to mix a few times but do not shake vigorously. 10. Place each tube back into its appropriate temperatures for 5 minutes (NO CAPS). 11. Label 3 new glucose strips “H” for Hot, “C” for Cold, and “RT” for Room Temperature. 12. Take glucose test strip “H” and dip it into tube HL (just enough to get the test pad wet). Immediately

remove the test strip and lay it down on the paper towel. 13. Take glucose test strip “C” and dip it into tube CL (just enough to get the test pad wet). Immediately

remove the test strip and lay it down on the paper towel. 14. Take glucose test strip “RT” and dip it into tube RTL (just enough to get the test pad wet). Immediately

remove the test strip and lay it down on the paper towel. 15. Wait 3 minutes and record color of the test strip and corresponding glucose amount in Table 3. 16. After completing the experiment, as a post-laboratory activity you will generate a line graph that shows

the dependence of enzyme activity on temperature. Be sure to put a title on the graph and label the axes.


Table 3: Temperature and Glucose Results Temperature Condition

Actual Temperature (°C)

Color of strip

Relative amount of glucose (0-3000mg/dl) after adding lactase

Ice

Room temperature

Hot water

Analysis Questions– Part 3

1. Which temperature represents the control? Why?

____________________________________________________________________________

2. What is the optimum temperature for Lactex®? How do you know?

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________

3. Explain the difference in enzyme activity at varying temperatures. What happened to the enzyme?

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________

4. Do you think it possible to recover the enzyme activity from the ones treated to extreme temperature

conditions? Why or why not? ____________________________________________________________________________

____________________________________________________________________________


Extension Activities Elaborate on Concepts

1. Using the graph, write a brief paragraph comparing the similarities and differences in the three

populations in terms of normal lactose tolerance.

Normal Lactose Tolerance by Ethnicity

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18

Age in Years

% N

orm

al L

acto

se T

oler

anc

Peruvian Mestizo U.S. CaucasianAfrican Americans

2. Using the data in the chart, construct a graph. Be sure to follow customary rules for graphing, including an appropriate title and labeled axes. Put the independent variable on the x axis and the dependent variable on the y axis. Then write a paragraph comparing the similarities and differences in the three populations in terms of normal lactose tolerance.

Normal Lactose Tolerance in U.S. Caucasian and African American Children and Peruvian Mestizo Children (in %)

Age in Years Caucasian African American Peruvian 2 No data No data 65 4 100 100 25 6 95 80 14 8 85 65 13 10 85 55 10 12 84 50 9 14 83 45 8 16 84 38 5 18 88 33 4

3. Propose a solution to the problem of school-age children in Baltimore City needing calcium and not making use of the milk provided with school lunches (due to their symptoms of lactose intolerance). Write this up in the form of a letter to the principal.

Extension Activities Page 18

4. While on a field trip with your biology class to visit a local farm, you accidentally become separated from your classmates and miss lunch. After a while, you get so hungry that you pull up some grass and eat it. Shortly afterward, you were reunited with your classmates. On the bus ride home a few hours later, you developed terrible cramps and gas and nobody wanted to sit near you. You don’t understand what happened. You see the cows, sheep, and goats eating grass, and they are mammals just like you. You asked your teacher about it, and she said that many herbivorous animals have compartmentalized stomachs that contain certain bacteria and protozoa. She also mentioned that humans don’t have the ability to digest cellulose, a carbohydrate found in the cell walls of plants.

a. In terms of enzymes, explain why eating the grass caused you to have digestive problems. _______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

b. Explain the role of bacteria and the protozoa in the stomachs of herbivorous mammals such

as cows, sheep, and goats. What do you think would happen to these animals if they did not have this symbiotic relationship with the bacteria and the protozoa?

_______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

5. Discuss in terms of evolution why the ability to produce lactase seems to be universal among humans for the first two years of life and diminishes for many groups of people after that.

6. Have students write up a package insert for their new Lactex® product which would give the

directions on how to take the tablets. This would include information regarding the types of foods you can ingest with the tablets (have the students consider acidic types of foods, basic or alkaline things such as antacid medicines, and also consider the temperatures of the food products to be taken with the tablets).


Answers to Extension Activities: 1. Similarities:

• All three groups show relatively high lactose tolerance at the earliest ages for which data is available.

• All three groups show a decrease in tolerance with age. (Note: mestizo refers to offspring of European and S. American Indian parents) Differences: • Peruvian mestizo children show the most rapid decrease in tolerance, being less than 20 %

tolerant by age 4, while the two other groups still show nearly 100% tolerance. • U.S. Caucasian children maintain an 80+ % tolerance to age 18 while African Americans

decrease steadily to less than 40%. • Although U.S. Caucasian children show a decrease after the age of 4, they actually show a

slight increase after the age of 16.

2. The student graphs should reflect the trends described in #1 above and the paragraph should include the information given for #1 above.

3. The letter should be written in standard business letter format. The letter should address the fact

that the majority of students in the Baltimore City Public School system are African Americans and thus a high percentage (maybe up to 70%) is likely to be lactose intolerant. The letter should propose a solution such as offering calcium fortified orange juice as an alternative to the milk or providing lactase supplemented milk.

4. a) Since humans cannot digest cellulose, this carbohydrate passes into the large intestine where

resident bacteria break down the cellulose and releases gas. b) The bacteria and protozoa that live in the stomachs of herbivorous mammals such as cows and sheep are able to digest the cellulose of the grass. The mammals can then absorb the glucose end product to use for energy. The bacteria and protozoa, in turn, get an appropriate supply of food and a place to live safe from predators. If the mammals did not have these symbiotic organisms living in their stomachs, they would be more similar to humans and not be able to utilize the cellulose in the plant cell walls.

5. Students should discuss the usefulness of the lactase enzyme by breast-fed infants and how this

would contribute to the survival rate of these children. As infants are weaned, the enzyme’s activity seems to decrease universally. The exceptions are those areas of the world where mammals were domesticated for providing milk as a source of nourishment for all segments of the population. The discussion should include the evolutionary advantage of maintaining functioning lactase enzymes in humans in these regions and the possibility that having this enzyme was a selective advantage that would be passed on genetically from one generation to the next.


Selected Response Questions Evaluation

Multiple Choice Questions 1. The enzyme _______ can break down _______ into _______ and galactose.

a. lactose, glucose, lactase b. lactase, lactose, glucose c. glucase, lactase, lactose d. lactase, glucose, lactose

2. Enzymes are _______ molecules which _______ specific chemical reactions.

a. carbohydrate, inhibit b. lipid, speed-up c. protein, speed-up d. nucleic acid, inhibit

3. The ability of an enzyme to function is influenced by the

a. indicator b. temperature c. enzyme shape d. both b and c

4. The presence of glucose can be shown by

a. a glucose test strip turning from yellow to green b. a lactose test strip turning from green to yellow c. a sample of milk turning from blue to yellow d. a sample of milk turning from blue to red

5. Which of the following groups of people retain into adulthood, with the largest percent, the

natural ability to digest lactose a. Asians b. Africans c. Latinos d. Northern Europeans

Answers for the Multiple Choice Questions 1. b 2. c 3. d 4. a 5. d


Short Answer Questions 1. At which temperature did the Lactex® tablets work best to break down the lactose in the milk—

freezing, room temperature, or hot? What does this suggest about how effective the Lactex® tablets would be for someone who likes ice cream compared with someone who drinks milk?

2. Look at the following graph showing the reaction rates of two enzymes.

Effect of pH on Enzyme Activity

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

pH

Rea

ctio

n R

ate

(mg/

sec)

Enzyme A

Enzyme B

Which enzyme works better in an alkaline environment? _______________________________

a. At which pH do the two enzymes work equally well? _____________________________ b. What is the optimum pH for Enzyme A?

_______________________________________________________________________ c. Describe the overall reaction rate pattern shown by both enzymes.

_______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

Answers for the Short Answer Questions 1. The enzyme worked best at room temperature, which is closest (of the temperatures used) to that

of body temperature. It would not work as well with the cold ice cream in which the enzyme is inactive, but not denatured. At hot temperatures, the protein enzyme molecule is denatured (the enzyme loses its specific 3-D structure).

2. a) Enzyme B

b) pH 5 c) pH 3 d) Both show a bell-shaped curve with a low reaction rate at the lower and upper levels of their ranges and an optimum rate approximately midway between each of their two extremes.


Bioscience… Real Jobs, Real People Post-Laboratory Activity Bioscience: Real Jobs, Real People follows four high school students to various Maryland bioscience companies on “Bioscience Career Day.” During the nine-minute film students interview scientists who work in a broad range of careers such as research, development, management, and manufacturing. Not only are their day to day responsibilities explained but each scientist also discusses universal truths about pursuing a career in the industry.

This video and an accompanying lesson plan are included in the loaner lab kit, and are to be returned. Additional copies of the video can be obtained by requests made through the MdBio web site and additional lesson plans can also be downloaded from the web site (http://www.mdbiolab.org).

Post-Laboratory Activity Page 23

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