Vocabulary: Objectives: Materials: Students will:

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1 Enzymes and Their Functions Authors: Jeisa Pelet and Carolyn Wilczynski Date Created: Subject: Living Environment Level: High School Standards: New York Sate Learning Standards Living Environment ( Standards 1: Analysis, Inquiry and Design Standards 4: Physical Setting and Living Environment Schedule: 5 periods of 45-minutes and 2 periods of 90-minutes Objectives: Students will learn about enzymes and how their activities are affected by different environmental factors. Following an introductory activity, students will conduct an experiment using amylase (enzyme) and starch (substrate) as an example. Students will: Understand the concept of enzymes, chemical reactions, catalysts and substrates. Correlate locks and keys with enzymes and substrates. Quantify the production of glucose from amylase/starch reaction in time. Develop an experimental design based on different factors affecting enzyme activity. Develop a problem statement and hypothesis, collect and analyze data, arrive at conclusions and complete a lab report. Vocabulary: Enzymes Chemical reaction Materials: For Each Pair: Part 1: Locks and keys Part 2 and 3 (each): 600 ml Beaker Dialysis tubing (~ 3cm width, molecular weight cutoff of 12,000 to 14,000 MW, Carolina) (15 cm) Starch (3 g) Amylase (1 g) Benedict s solution (3 ml) Pipettor (P1000) Pipette tips (1 ml) Transfer pipettes (2) 2 ml Eppendorf tubes (7) Floating eppendorf tube holder Catalyst Substrate Tube holder Plastic cuvettes (7) Cuvette holder Waste container Magnetic stir bar (3.5-5 cm) Dialysis clips (2) Timer Stir plate Stir/heating plate For Each Student: Safety goggles Gloves Activity sheets For the Class: Spectrophotometer Small centrifuge Vortex Safety: Benedicts solution should be handled with care. Students should wear safety goggles, lab coats, and gloves when working with Benedicts solution and hot water. If possible, use heat resistant gloves when handling hot water.

2 Introduction To study how the enzymes act upon the substrates, we will use amylase and starch as an enzyme and a substrate, respectively. Amylase converts polysaccharides into monosaccharides. Polysaccharides are long chains of sugars attached together, while monosaccharides are single sugar molecules. In the case of starch (a polysaccharide), amylase will break it down into glucose (monosaccharide) as illustrated below. amylase starch glucose If amylase and starch are placed inside a dialysis tubing, the amylase will immediately begin to break the starch down into glucose. As the glucose forms from the enzyme activity, it will diffuse out through the membrane because it is small enough to fit through the mesh of the membrane. However, the amylase and the starch will stay inside the dialysis tubing because these molecules are too large to fit through the mesh. By detecting the amount of glucose outside the dialysis tubing over a period of time, we can study the rate (how fast or how slow) of the enzyme activity. This lesson on enzymes consists of three parts: Part 1 (Day 1 2): Enzymes and Their Functions Lock-and-Key Activity Part 2 (Day 3 4): Enzyme Action: The Breakdown of Starch into Glucose Part 3 (Day 5 7): Inquiry-based Activity on Enzymes All three parts can be performed sequentially. However, if time is a constraint, each part can be done independently, except for Part 3 which requires Part 2 as an introduction. Student s Pre-requisites: It is recommended that the following Lab is completed before this lesson: New York State Education Department (NYSED) "Diffusion Through a Membrane". A laboratory activity produced by the State Education Department for use in fulfilling part of the laboratory requirement for the Regents Examination in Living Environment ( Page2 If the 'Diffusion Through a Membrane' lab is not available, the main concepts covered by this lab can be found in the Living Environment Core Curriculum:

3 Science Content: What are Enzymes? Enzymes are compounds that assist chemical reactions by increasing the rate at which they occur. For example, the food that you eat is broken down by digestive enzymes into tiny pieces that are small enough to travel through your blood stream and enter cells. Enzymes are proteins that are found in all living organisms. Without enzymes, most chemicals reactions within cells would occur so slowly that cells would not be able to work properly. Enzymes function as catalysts. Catalysts accelerate the rate of a chemical reaction without being destroyed or changed. They can be reused for the same chemical reaction over and over, just like a key can be reused to open a door many times. Enzymes are generally named after the substrate affected, and their names usually end in - ase. For example, enzymes that break down proteins are called proteases. While lipases break down lipids, carbohydrases break down carbohydrates. The compounds that enzymes act upon are known as substrates. The substrate can bind to a specific place in the enzyme called the active site. By temporarily binding to the substrate, an enzyme can lower the energy needed for a reaction to occur, thus making this reaction faster. The energy required for a chemical reaction to occur is known as the activation energy. Once the reaction between an enzyme and a substrate is complete, the substrate is changed to a product while the enzyme remains unchanged. The rate of the reaction between an enzyme and a substrate can be affected by different factors. Some of the factors that can affect enzyme activity are temperature, ph, concentration of the enzyme and concentration of the substrate. In living organisms, enzymes work best at certain temperatures and ph values depending on the type of enzyme. A Little More Information on Diffusion and Dialysis Membranes Page3 Molecules are in constant motion. So when there is a difference in concentration, or a concentration gradient of a specific particle, the particles will move toward the lower concentration in order to maintain an evenly distribution across the whole area. This movement of particles from higher concentration to lower concentration is known as diffusion. A dialysis membrane is a mesh-like material that will only allow certain particles of a specific size or smaller to pass or diffuse through. For example, if inside a dialysis tubing we place grains of rice and salt, the salt will diffuse out the tubing because it is small enough to move through the mesh of the membrane. The rice, however, will stay inside the tubing because it is too large to move through the mesh of the membrane. Kidneys are similar to a dialysis membrane. As blood passes through the kidneys, small molecules will be filtered out and eliminated from the body as urine.

4 Preparation: 1. Photocopy the Activity Sheets corresponding to each Part (per student). 2. Perform the following preparation steps for each Part accordingly. Part 1 (Day 1 2): Enzymes and Their Functions Lock-and-Key Activity Prepare two individual sets of 4 locks and 4 keys each (one per group). Set 1: Locks and keys will all have different shapes and sizes, and each key will open only one lock. Set 2: Locks and keys will all have different shapes and sizes, one key will open two locks, one key will open only one lock and two keys will not open any locks. Part 2 (Day 3 5): Enzyme Action: The Breakdown of Starch into Glucose A. Glucose calibration curve A calibration curve of glucose concentration with absorbance should be performed. This calibration curve is done by preparing various solutions of different known concentrations of glucose with fixed amount of Benedicts solution. The absorbance of each sample is then detected using a spectrophotometer, and a plot of absorbance (y-axis) vs. glucose concentration (x-axis) is generated. A linear regression of this data will provide a slope (m) and a y-intercept (b) values according to the following equation: The m and b values will be used for the calculation of glucose concentration in the unknown samples. Detailed instructions on how to perform the calibration curve can be found in Supplementary Information. Alternatively, if time is a constraint, the values of m = and b = 0.59 can be used. B. Materials to be prepared prior to lab day 1. In a vial, weigh 3g of starch (one per group). 2. In a vial, weigh 1g of amylase (one per group). Note: If amylase is not going to be used immediately, place in the fridge after weighing and do not dissolve with water until ready for the experiment. 3. Cut 15 cm of dialysis tubing (one per group). Part 3 (Day 4 7): Inquiry-Based Activity on Enzymes Page4 Part 3 requires the same materials and preparations as in Part 2. Additional materials (i.e. ph meter, sodium chloride (NaCl) solution, sodium hydroxide (NaOH) solution, ice) might be required depending on the experiment designed by the students.

5 Classroom Procedure: Part 1 (Day 1 2): Enzymes and Their Functions Lock-and-Key Activity Objective: The objective of this activity is to introduce the concept of enzymes and their functions through the lock-and-key model by using real locks and keys as an analogy. Students can be divided in groups of 2 or 3. Procedure Part 1.1: 1. Hand out the Activity Sheets Part 1 (page 3 7) (per student). 2. To each group of students, provide Set 1 of locks/keys. 3. Ask each group to try all the keys with all the locks and to answer the following questions: a. Were you able to open all locks? b. Do all keys open all locks? c. Can 1 key open more than 1 lock? d. Can you open the same lock with the same key more than once? e. Do all keys have the same shape? 4. Collect Set 1 of locks/keys and continue with Part 1.2. Procedure Part 1.2: 1. Ask each group of students to make 3 predictions (from previous observations) about a new set of locks/keys to be given (Set 2). 2. To each group provide Set 2 of locks/keys. 3. Ask the students to test their predictions and to say if whether or not each prediction was valid based on their results. 4. Ask the students to make 3 additional observations about Set 2 of locks/keys. 5. Ask the students to make comparisons between enzymes/substrates and keys/locks (3 similarities and 3 differences). 6. Ask the students to share their ideas and write them on the blackboard. 7. Allow students to complete the questions in the Activity Sheets Part 1 (B). Page5 After this activity, the lock-and-key model can be correlated to enzymes and substrates. Different properties on enzymes/substrates can be addressed and compared to keys/locks. For example, enzymes (keys) are reusable, specific, have different shapes, produce a change to a substrate (lock), and fit in an active site with a substrate. It would also be important to talk about factors that affect enzyme activity, such as temperature, ph and enzyme/substrate concentration.

6 Part 2 (Day 3 4): Enzyme Action: The Breakdown of Starch into Glucose Objective: The objective of this activity is to perform an experiment with an enzyme (amylase) and a substrate (starch), and to understand how enzymes work. Each group of students will set up the same control experiment (6 time points) for minutes (depending on the time available). The amount of glucose produced from the amylase/starch reaction will be quantified in time. Data will be compared among students. Students should be divided in groups of 2 or 3. Procedure Part 2.1: Collecting Samples (Day 3) 1. Provide the following materials to students (per group): a. Beaker (600mL) j. Pipette tips (1 ml) b. Stir plate k. Waste container c. Stir bar l. Transfer pipette (2) d. Graduated cylinder (500 ml) m. 2 ml Eppendorf tubes (7) e. 15 cm Dialysis tubing (pre-cut) n. Tube holder f. Pre-weighted starch (3g) o. Timer g. Pre-weighted amylase (1g) p. Vortex (1 per class) h. Water (~510 ml) q. Activity Sheets Part 2 (page 8-14) i. Pippetor (P1000) (per student) Page6 The following are the steps for collecting samples (same as in Activity Sheets, Page 10): 2. Select the times at which samples will be collected. Note: Recommended times are 0, 5, 10, 20,30 and 40 minutes. 3. Write these times (minutes) in Table Label 7 eppendorf tubes with numbers from 1 to 7 and your initials. 5. Using a graduated cylinder, measure 500 ml of water and place it in the beaker. 6. Remove 1.5 ml of the water from the beaker and place it in tube Dissolve 3 g of starch by adding 5 ml of water to the vial. Note: a white cloudy solution should be seen. 8. Shake or vortex the starch solution. 9. Dissolve 1 g of amylase by adding 5 ml of water to the vial. Note: A brown solution should be seen. 10. Shake or vortex the amylase solution. 11. Seal the bottom of the dialysis tubing by wrapping one of the ends around a stir bar and clipping this end with a dialysis clip. 12. Use a transfer pipette to add all of the starch solution into the dialysis tubing.

7 13. Use another transfer pipette to add all of the amylase solution into the dialysis tubing. 14. Clip the other end (top part) of the dialysis tubing with a dialysis clip. 15. Insert the sealed dialysis tubing in the beaker with water. 16. Start the timer. 17. Set the stir dial in the stir plate to the lowest setting. 18. At each specific time (see Table 1), remove 1.5mL of the water from the beaker and place it in a 2 ml eppendorf tube labeled with the number corresponding to that time (tubes 2 6). 19. To tube 7, add 1.5 ml of water. Additional Notes: Note 1: if pippettors are not available, use graduated plastic pipettes to collect the samples. Note 2: The amylase will form a brown layer on the top while the starch will form a white layer on the bottom of the dialysis tubing. As time passes, the brown layer (amylase) will increase as the white layer (starch) will decrease. Note 3: If the data analysis is going to be done on a different day, the samples collected by the students can be stored in the freezer. Note 4: A negative control experiment can be performed where only starch is placed in the dialysis tubing (no amylase) and Benedict s solution is used to detect for the presence of glucose after minutes. Procedure Part 2.2: Analyzing the Data (Day 4) 1. Provide the following materials to students (per group): a. Beaker (600 ml) i. Waste container b. Stir/heating plate j. Plastic cuvettes (7) c. Stir bar k. Cuvette holder d. Samples from Part 2.1 l. Floating eppendorf tube holder e. Benedict s solution (~ 2 ml) m. Timer f. Water (~300 ml) n. Small centrifuge (1 per class) g. Pippettor (P1000) o. Vortex (1 per class) h. Pipette tips (1 ml) p. Spectrophotometer (1per class) Page7 The following are the steps for analyzing the data (same as in Activity Sheets, Page 11): 2. Start a stirring water bath at ~80 90 C using a heating/stir plate. Note: Do not allow water to boil. 3. Turn on the spectrophotometer. Note: General instructions on how to use a spectrophotometer are in Supplementary Information.

8 4. Label 7 plastic cuvettes with numbers from 1 to To each sample (1 6), except tube 7, add 0.3 ml of Benedicts solution. Note: tube 7 will have only water. 6. Shake or vortex each tube for 5 seconds. 7. Place all the tubes in a floating eppendorf tube holder. 8. Place the floating eppendorf tube holder in the heating water bath. 9. Start the timer. 10. Place a stir bar in the water bath and set the stir dial in the plate to the lowest setting. 11. After 10 minutes, remove the samples from the water bath. Note: Caution! Floating eppendorf tube holder and tubes will be very hot. Remove the tube holder using the tab that is above the water level. If possible, use heat resistant gloves. 12. Allow the samples to cool down to room temperature (~ 3 minutes). 13. Centrifuge the tubes for 3 minutes. 14. Gently remove your samples from the centrifuge and place them in the tube rack. Do not shake them. 15. Without disturbing the solution in your sample, use a pippetor to remove 1 ml of the liquid and place it in the plastic cuvette with the same number. Note: Do not to touch the bottom of the tube where the red precipitate may be present. 16. Set the spectrophotometer to 735 nm. 17. Use cuvette 7 (only water) to set a reference for the spectrophotometer. 18. Obtain the absorbance of each sample (cuvettes 1 6). 19. Write down the absorbance of each sample in Table Obtain the m and b values from the glucose calibration curve. 21. Calculate the glucose concentration for each sample, and complete Table 2. Note: Glucose concentration is calculated with:, where y is the absorbance, and m and b are from the glucose calibration curve. 22. With the data in Table 2, plot glucose concentration (y-axis) vs. time (x-axis). The following plot of glucose concentration vs. time should be expected: Glucose Concentration Vs. Time 0.2 Page8 Glucose Concentration (mg/ml) Time (minutes)

9 Part 3 (Day 5 7): Inquiry-Based Activity on Enzymes Objective: The objective of this activity is to understand how different factors can affect enzyme activity. Each group of students will select an environmental factor that they would like to analyze and they will design their own experiment using the knowledge gained from the previous activities. This experiment will answer a question based on the effect of the selected factor on enzyme activity. Students can be divided in groups of 2 or 3. Procedure Part 3.1: Designing an Experiment (Day 5) 1. Hand out Activity Sheets Part 3 (page 14 20) (per student). 2. Ask the students to write down in the Lab Report two factors that they think will affect enzyme activity. 3. Allow the students to discuss these factors among their group and to pick one factor to study. 4. Allow the students to design an experiment with their partner(s) that will answer a question based on the selected factor and enzyme activity, and to complete Part A and B of their Lab Report. Note 1: Examples of factors can be: temperature (higher or lower), ph (acid or basic) or enzyme/substrate concentration. (Make sure that you approve the experiment to be performed). Students can follow the basic procedure in Part 2.1 and 2.2 with adjustments depending on their experimental design. Procedure Part 3.2: Inquiry-Based Activity (Day 6 7) 1. Provide materials to students. This will depend on their experimental design (basic materials are those from Part 2.1 and 2.2). 2. Allow the students to set up and run their experiments, collect data, discuss their results and complete the rest of the Lab Report. Note: Students should use the data from Part 2 to compare to their results. An example of temperature effect on enzyme activity is as following: Page9 Glucose Concentration (mg/ml) Glucose Concentration Vs. Time (T=48 C) Time (minutes)

10 Supplemental Information: Glucose Calibration Curve: Materials: a. Stir plate b. Stir/heating plate c. Glucose (2 mg) d. Water e. Benedict s solution f. Pipettor (P1000) g. Pipette tips (1 ml) h. 2 ml Eppendorf tubes (8) i. Tube holder j. Plastic cuvettes (8) k. Cuvette holder l. Floating eppendorf tube holder m. Vortex n. Small centrifuge o. Spectrophotometer Procedure: 1. Start a stirring water bath at ~ C using a heating/stir plate. Note: Do not allow water to boil. 2. Turn on the spectrophotometer. 3. Weigh 2 mg of glucose and place it in an eppendorf tube or vial. 4. Dissolve the glucose with 2 ml of water to make a 1 mg/ml solution. 5. Label 8 eppendorf tubes (2 ml) with numbers from 1 to Add the following (from Table S1) to each tube with the corresponding number: Table S1 Tube # Glucose stock solution, 1 mg/ml (ml) Distilled water (ml) Benedicts solution (ml) Final concentration (mg/ml) Page10 7. Vortex each tube for 5 seconds. 8. Place all the tubes in a floating eppendorf tube holder. 9. Place the floating eppendorf tube holder in the heating water bath for 10 minutes.

11 10. Remove the tubes from the water bath and allow them to cool down to room temperature (~ 3 minutes). 11. Centrifuge the tubes for 3 minutes. 12. Without disturbing the solution in your sample, use a pippetor to remove 1mL of the liquid and place it in the plastic cuvette labeled with the same number. Note: Do not to touch the bottom of the tube where the red precipitate may be present. 13. Set the spectrophotometer to 735 nm. 14. Use cuvette 8 (only water) to set a reference for the spectrophotometer. 15. Obtain the absorbance of each sample (cuvettes 1 7). 16. Record the data in Table S2, and plot absorbance (y-axis) vs. glucose concentration (x-axis). Note: a linear plot with a negative slope should be observed. 17. Perform a linear trend (in excel) and obtain the slope (m) and y-intercept (b) values according to the following equation: Table S2 Tube # Final glucose concentration (mg/ml) Absorbance at 735 nm Slope (m) = y-intercept (b) = The following plot of glucose concentration with absorbance is expected (in this case, galactose was used as the monosaccharide): Page11 Absorbance (735 nm) E-16 Calibration Curve y = x R² = [Galactose] (mg/ml)

12 Instructions for using the Spectrophotometer: For Spectro Master (Model 415) spectrophotometer (other models may vary) 1. Turn on the spectrophotometer. Allow to warm for ~30 minutes. 2. Open the sample compartment and set the filter selector to the appropriate position depending on the desired wavelength range. 3. Use the dial on the right side to set the wavelength to 735 nm (shown in the front window). 4. Press Trans/Abs to select ABS in the front panel. 5. Insert cuvette with only water into the holder inside the sample compartment. The smooth side of the cuvette should be facing forward. 6. Close the lid. 7. Press 100%/T/0 A and wait for a reading to appear in the front panel. This reading should be Remove the cuvette from the holder inside the sample compartment and place the new cuvette into this holder. 9. Close the lid and wait for a reading to automatically appear in the front panel. Basic Materials for Part 2 and Part 3: Page12

13 Safety: Benedicts solution should be handled with care. It is advised that students protect their eyes and skin by wearing protecting goggles and gloves when working with Benedicts solution and hot water. If possible, use heat resistant gloves when working with hot water. Acknowledgements: Glucose quantification was adapted from the Cell Biology Technique Manual: Benedict s Test (Hendrix College Biology Department). Cornell BME NSF GK-12 program: DGE Thanks to Dr. Michael Shuler, Dr. Chris B. Schaffer, Dr. Shivaun Archer, Nevjinder Singhota and Dr. David Putnam. Cornell University, Ithaca NY Page13