Enzymes (B)
Enzymes (B) Objectives
Explain how enzymes function Describe the terms active site, substrate, and product Describe the effects of optimal conditions on enzyme activity Apply the scientific method to answer a question regarding enzyme activity
Background Information: Chemical reactions require activation in order to for the reaction to “go.” Without enough activation energy, the reaction cannot begin. Activation energy essentially increases the likelihood that the reactants will collide with enough energy to provide the impetus for the reaction to occur. In a chemistry lab, activation energy is most often achieved by adding heat and/or by increasing the concentration of the reactants, but can also be accomplished by increasing pressure. Alternatively, chemists can use a catalyst to facilitate the reaction. Catalysts are not consumed in a reaction, but facilitate a reaction, sometimes by supplying the activation energy required. Organisms require many reactions to maintain life, including metabolism, homeostasis, and for growth and tissue repair. Biological reactions are chemical reactions, and just like chemical reactions, require activation energy in order to proceed. Most organisms could not survive the increase in temperature or pressure to meet activation energy requirements for the reactions, and an increase in temperature would apply to the entire organism, thus initiating more reactions than are intended. Reactant and product concentrations are useful in controlling biological reactions in a system called negative feedback. However, the reactant quantities necessary to ensure the reaction will occur are often in excess of the organism’s needs or ability to manage. Enzymes, which are proteins that act as biological catalysts, are evolution’s answer to this need to initiate reactions within safe limits. Several factors influence an enzyme’s rate of reaction (how quickly the enzyme can facilitate a reaction). The term “optimal conditions” describes the ideal conditions needed to maximize the enzyme’s reaction rate. Exploration Questions: 1. If enzymes are biological catalysts, are they consumed in a reaction? Explain.
2. Would the body require different enzymes for different reactions or do you think a few enzymes facilitate all of the body’s reactions? Explain.
3. What factors, environmental or other, impact a protein’s function? Would these same factors
apply to enzyme activity? Explain.
4. Predict the optimal conditions for an enzyme found in the stomach. Explain your reasoning.
The Lab Scenario Please read the following email: Hi Kathy, My brother’s new baby was diagnosed with an amylase deficiency. He was upset when he talked about it, so I wasn’t able to get the whole story from him. In fact, I’m not sure he understood everything the doctor told him about amylase deficiency because he was so upset to hear the news. What I did understand is that amylase has something to do with the mouth and breaking down carbohydrates, but I don’t know more than that. I’d like to learn more so I can help him out. Since you’re in a biology lab for your internship, I thought you might be able to explain a bit more for me. What does amylase do? If the baby is deficient, maybe she still makes some amylase. Are there conditions that change amylase’s effectiveness, or is it always equally effective? If amylase is connected to carbohydrates, does she need to worry about other food groups like protein and fats? Thanks for any help you can give me! Kaylie 1. What questions does Kaylie want answered? Make a prediction or state a hypothesis regarding the answer to each question. Question asked Prediction or Hypothesis
Part I. What does amylase actually do? We will test amylase against each of the major biological molecules in foods, carbohydrates, fats (lipids), and proteins. Review the food label below to learn a little about each of these biological molecules. Note that fats are divided into two subgroups, carbohydrates are divided into two subgroups, and proteins are in a single category. Fats: While this label only shows two subcategories, dietary fats can be artificial (trans) or naturally occurring saturated or unsaturated fats. Unsaturated fats have the most health benefits while trans fats are the least healthy. All of these fats will react identically in a lipid test. Carbohydrates: Carbohydrates appear as dietary fiber and sugars. Dietary fiber is a complex carbohydrate which humans cannot digest (i.e., cellulose). Dietary fiber helps improve digestive health and can help to prevent a variety of health concerns such as diabetes, weight gain, and some cancers. The “Sugars” listing on a nutrition label includes both simple sugars and digestible complex carbohydrates, such as starch. These sugars provide the energy necessary to support life. Benedict’s test detects simple sugars or simple carbohydrates. The iodine test detects starch, not dietary fiber. Proteins: Proteins in the diet are not subdivided. A test is available to detect protein. NOTE: Directions are provided for the Benedict’s Test and Iodine Test. See the Biological Molecules Lab for directions for the lipid test and a protein test. Consult your instructor to discuss if you decide you want to run a lipid or protein test.
Available Materials Spot plates (three per group) Test tubes Test tube racks (three per group) IKI (Iodine) solution Benedict’s solution Beaker and hot plate (for boiling water bath) Test tube tongs
Thermometers (three per group) 37°C incubator Refrigerator Bench top pH buffer solutions (pH 3, pH 7, pH 11) pH test strips Starch solution Glucose solution Lipid solution Protein solution Deionized water Paper cups for collecting salivary amylase Procedure for Benedict’s Test (simple sugars) Procedure for Iodine Test (complex carbohydrates) Procedure for Collecting Salivary Amylase
Procedural Notes: Procedure for Collecting Salivary Amylase 1. To collect the salivary amylase, you will collect your saliva in the small paper cup provided. 2. The saliva can be provided by a single group member or by multiple group members. 3. Do NOT chew gum while doing this. Do NOT eat or drink anything but water while collecting the saliva. 4. You will need about 1 mL of saliva for each test tube tested. Procedure for Iodine Test for Starch You will use a spot plate to perform the iodine test for starch. Be sure to label each well of the spot plate prior to placing any sample in the wells! 1. Place a drop from the test solution into the appropriate well of the spot plate. 2. Add one drop of iodine to the test solution in the spot plate. 3. Monitor the color: an amber color is a negative result for starch, while a bluish-black color is a positive result for starch. Procedure for Benedict’s Test for Simple Sugars You will use test tubes to perform the Benedict’s test for simple sugars. Be sure to label each test tube prior to placing any sample in the tubes! 1. Turn on the water baths and bring the water to a boil. 2. Place 0.5 mL of solution from each sample stock tube into the appropriately labeled test tube. 3. Add 0.5 mL of Benedict’s solution to each test tube. 4. Boil tubes in water bath for 3 minutes.
5. Record results. No change in color (remains blue) is a negative result for simple sugars. A green color is a weak positive result. A yellow color is a moderately positive result. A brickred color is a strong positive result for simple sugars.
Part I. Experimental Design (What does amylase do?) As you think about the steps involved in this investigation, you might consider the following: Why might you need an incubation time between adding amylase and testing for sugars and starches? Should you test each substance for starch at the before treatment, after treatment, or both before and after treatment. Should you test each substance for simple sugars before treatment, after treatment, or both before and after treatment? When adding reagents to a test tube, must you be consistent in each test tube with the amounts added?
1. Identify each element of for this investigation in the space provided: a. Hypothesis:
b. Independent Variable(s):
c. Dependent Variable(s):
d. Control Group(s):
e. Experimental Group(s):
f. Procedure Review the un-sequenced steps and put them in a logical order. If required, check your sequence with your instructor. Begin when instructed.
Note: You are responsible for cleaning all equipment (spot plate, test tubes, etc.) today!
2. Run your investigation and record your data and observations in the space below.
Part I Conclusions: What does amylase do? What can you tell Kaylie about amylase’s action and whether she needs to worry about other biological molecules? Be sure to use evidence from your investigation to support your answer. 3. Does amylase have any action on lipids? How did you know?
4. Does amylase have any action on proteins? How did you know?
5. Does amylase have any action on simple carbohydrates? How did you know?
6. Does amylase have any action on starch (complex carbohydrates)? How did you know?
Part II. What environmental conditions impact amylase’s activity? Now that we know amylase’s action, we can more effectively design an investigation to test for the environmental conditions that will impact its activity. We have materials available to test for amylase’s activity in three different temperatures and three different pH’s. Select one of the two
environmental conditions (pH or temperature) and collect the un-sequenced protocol strips for your environmental condition. As a class, both conditions must be evaluated, so check with other groups at your work table and make sure both pH and temperature are tested by different groups at your work table. Experimental Design, pH OR Temperature (Please circle which you are investigating.) As you sequence your steps for this investigation, you might consider: Should you treat the amylase with pH or temperature changes before adding to your test solution, or should you treat the test solution then add amylase? Will your results be the same, depending on which you do? Should you use the same ratio of test solution to amylase in each test tube?
1. Identify each element for this investigation in the space provided: Hypothesis:
Independent variable(s):
Dependent variable(s):
Control group(s):
Experimental group(s):
2.
Sequence the steps of the procedure and record your steps in the space below. Note the space for observations and notes to be completed while you work through your steps. Procedural Steps Observations/Notes
Results: Your Data:
Class Data Table: Treatment
Time 0 min (before treatment) Starch Simple sugar (+ or -) (+ or -)
Time 5 min (after treatment) Starch Simple Sugar (+ or -) (+ or -)
Refrigerator 37°C Incubator Boiled pH 3 pH 7 pH 11
Part II Conclusions: What environmental conditions impact amylase’s activity? Share your results with Kaylie. 1. Does temperature affect amylase activity? If so, at what temperature is amylase most effective? Does this make sense, given where amylase is found in the body? Explain using evidence from your investigation to support your answer.
2. Does pH affect amylase activity? If so, at what pH is amylase most effective? Does this make sense, given where amylase is found in the body? Explain using evidence from your investigation to support your answer.
Enzymes Lab Report Name: ____________________________________ Answer the following questions: 1. Make a table showing the results of your tests for optimal temperature. Based on your results, what is the optimal temperature for salivary amylase? What evidence that you collected supports this conclusion? What was your hypothesis? Did your results support your hypothesis, or if your hypothesis was not supported, did you still learn the optimal temperature range? (4 points)
2. Make a table showing the results of your tests for optimal pH. Based on your results, what is the optimal pH for salivary amylase? What evidence that you collected supports this conclusion? What was your hypothesis? Did your results support your hypothesis, or if your hypothesis was not supported, did you still learn the optimal pH range? (4 points)
3. When conducting laboratory experiments, it is useful to include positive and negative controls to verify that your experimental results are valid. What would be used for positive and negative controls for the Benedict’s assay? For the iodine test? (2 points)
Explain how enzymes function Describe the terms active site, substrate, and product Describe the effects of optimal conditions on enzyme activity Apply the scientific method to answer a question regarding enzyme activity
Background Information: Chemical reactions require activation in order to for the reaction to “go.” Without enough activation energy, the reaction cannot begin. Activation energy essentially increases the likelihood that the reactants will collide with enough energy to provide the impetus for the reaction to occur. In a chemistry lab, activation energy is most often achieved by adding heat and/or by increasing the concentration of the reactants, but can also be accomplished by increasing pressure. Alternatively, chemists can use a catalyst to facilitate the reaction. Catalysts are not consumed in a reaction, but facilitate a reaction, sometimes by supplying the activation energy required. Organisms require many reactions to maintain life, including metabolism, homeostasis, and for growth and tissue repair. Biological reactions are chemical reactions, and just like chemical reactions, require activation energy in order to proceed. Most organisms could not survive the increase in temperature or pressure to meet activation energy requirements for the reactions, and an increase in temperature would apply to the entire organism, thus initiating more reactions than are intended. Reactant and product concentrations are useful in controlling biological reactions in a system called negative feedback. However, the reactant quantities necessary to ensure the reaction will occur are often in excess of the organism’s needs or ability to manage. Enzymes, which are proteins that act as biological catalysts, are evolution’s answer to this need to initiate reactions within safe limits. Several factors influence an enzyme’s rate of reaction (how quickly the enzyme can facilitate a reaction). The term “optimal conditions” describes the ideal conditions needed to maximize the enzyme’s reaction rate. Exploration Questions: 1. If enzymes are biological catalysts, are they consumed in a reaction? Explain.
2. Would the body require different enzymes for different reactions or do you think a few enzymes facilitate all of the body’s reactions? Explain.
3. What factors, environmental or other, impact a protein’s function? Would these same factors
apply to enzyme activity? Explain.
4. Predict the optimal conditions for an enzyme found in the stomach. Explain your reasoning.
The Lab Scenario Please read the following email: Hi Kathy, My brother’s new baby was diagnosed with an amylase deficiency. He was upset when he talked about it, so I wasn’t able to get the whole story from him. In fact, I’m not sure he understood everything the doctor told him about amylase deficiency because he was so upset to hear the news. What I did understand is that amylase has something to do with the mouth and breaking down carbohydrates, but I don’t know more than that. I’d like to learn more so I can help him out. Since you’re in a biology lab for your internship, I thought you might be able to explain a bit more for me. What does amylase do? If the baby is deficient, maybe she still makes some amylase. Are there conditions that change amylase’s effectiveness, or is it always equally effective? If amylase is connected to carbohydrates, does she need to worry about other food groups like protein and fats? Thanks for any help you can give me! Kaylie 1. What questions does Kaylie want answered? Make a prediction or state a hypothesis regarding the answer to each question. Question asked Prediction or Hypothesis
Part I. What does amylase actually do? We will test amylase against each of the major biological molecules in foods, carbohydrates, fats (lipids), and proteins. Review the food label below to learn a little about each of these biological molecules. Note that fats are divided into two subgroups, carbohydrates are divided into two subgroups, and proteins are in a single category. Fats: While this label only shows two subcategories, dietary fats can be artificial (trans) or naturally occurring saturated or unsaturated fats. Unsaturated fats have the most health benefits while trans fats are the least healthy. All of these fats will react identically in a lipid test. Carbohydrates: Carbohydrates appear as dietary fiber and sugars. Dietary fiber is a complex carbohydrate which humans cannot digest (i.e., cellulose). Dietary fiber helps improve digestive health and can help to prevent a variety of health concerns such as diabetes, weight gain, and some cancers. The “Sugars” listing on a nutrition label includes both simple sugars and digestible complex carbohydrates, such as starch. These sugars provide the energy necessary to support life. Benedict’s test detects simple sugars or simple carbohydrates. The iodine test detects starch, not dietary fiber. Proteins: Proteins in the diet are not subdivided. A test is available to detect protein. NOTE: Directions are provided for the Benedict’s Test and Iodine Test. See the Biological Molecules Lab for directions for the lipid test and a protein test. Consult your instructor to discuss if you decide you want to run a lipid or protein test.
Available Materials Spot plates (three per group) Test tubes Test tube racks (three per group) IKI (Iodine) solution Benedict’s solution Beaker and hot plate (for boiling water bath) Test tube tongs
Thermometers (three per group) 37°C incubator Refrigerator Bench top pH buffer solutions (pH 3, pH 7, pH 11) pH test strips Starch solution Glucose solution Lipid solution Protein solution Deionized water Paper cups for collecting salivary amylase Procedure for Benedict’s Test (simple sugars) Procedure for Iodine Test (complex carbohydrates) Procedure for Collecting Salivary Amylase
Procedural Notes: Procedure for Collecting Salivary Amylase 1. To collect the salivary amylase, you will collect your saliva in the small paper cup provided. 2. The saliva can be provided by a single group member or by multiple group members. 3. Do NOT chew gum while doing this. Do NOT eat or drink anything but water while collecting the saliva. 4. You will need about 1 mL of saliva for each test tube tested. Procedure for Iodine Test for Starch You will use a spot plate to perform the iodine test for starch. Be sure to label each well of the spot plate prior to placing any sample in the wells! 1. Place a drop from the test solution into the appropriate well of the spot plate. 2. Add one drop of iodine to the test solution in the spot plate. 3. Monitor the color: an amber color is a negative result for starch, while a bluish-black color is a positive result for starch. Procedure for Benedict’s Test for Simple Sugars You will use test tubes to perform the Benedict’s test for simple sugars. Be sure to label each test tube prior to placing any sample in the tubes! 1. Turn on the water baths and bring the water to a boil. 2. Place 0.5 mL of solution from each sample stock tube into the appropriately labeled test tube. 3. Add 0.5 mL of Benedict’s solution to each test tube. 4. Boil tubes in water bath for 3 minutes.
5. Record results. No change in color (remains blue) is a negative result for simple sugars. A green color is a weak positive result. A yellow color is a moderately positive result. A brickred color is a strong positive result for simple sugars.
Part I. Experimental Design (What does amylase do?) As you think about the steps involved in this investigation, you might consider the following: Why might you need an incubation time between adding amylase and testing for sugars and starches? Should you test each substance for starch at the before treatment, after treatment, or both before and after treatment. Should you test each substance for simple sugars before treatment, after treatment, or both before and after treatment? When adding reagents to a test tube, must you be consistent in each test tube with the amounts added?
1. Identify each element of for this investigation in the space provided: a. Hypothesis:
b. Independent Variable(s):
c. Dependent Variable(s):
d. Control Group(s):
e. Experimental Group(s):
f. Procedure Review the un-sequenced steps and put them in a logical order. If required, check your sequence with your instructor. Begin when instructed.
Note: You are responsible for cleaning all equipment (spot plate, test tubes, etc.) today!
2. Run your investigation and record your data and observations in the space below.
Part I Conclusions: What does amylase do? What can you tell Kaylie about amylase’s action and whether she needs to worry about other biological molecules? Be sure to use evidence from your investigation to support your answer. 3. Does amylase have any action on lipids? How did you know?
4. Does amylase have any action on proteins? How did you know?
5. Does amylase have any action on simple carbohydrates? How did you know?
6. Does amylase have any action on starch (complex carbohydrates)? How did you know?
Part II. What environmental conditions impact amylase’s activity? Now that we know amylase’s action, we can more effectively design an investigation to test for the environmental conditions that will impact its activity. We have materials available to test for amylase’s activity in three different temperatures and three different pH’s. Select one of the two
environmental conditions (pH or temperature) and collect the un-sequenced protocol strips for your environmental condition. As a class, both conditions must be evaluated, so check with other groups at your work table and make sure both pH and temperature are tested by different groups at your work table. Experimental Design, pH OR Temperature (Please circle which you are investigating.) As you sequence your steps for this investigation, you might consider: Should you treat the amylase with pH or temperature changes before adding to your test solution, or should you treat the test solution then add amylase? Will your results be the same, depending on which you do? Should you use the same ratio of test solution to amylase in each test tube?
1. Identify each element for this investigation in the space provided: Hypothesis:
Independent variable(s):
Dependent variable(s):
Control group(s):
Experimental group(s):
2.
Sequence the steps of the procedure and record your steps in the space below. Note the space for observations and notes to be completed while you work through your steps. Procedural Steps Observations/Notes
Results: Your Data:
Class Data Table: Treatment
Time 0 min (before treatment) Starch Simple sugar (+ or -) (+ or -)
Time 5 min (after treatment) Starch Simple Sugar (+ or -) (+ or -)
Refrigerator 37°C Incubator Boiled pH 3 pH 7 pH 11
Part II Conclusions: What environmental conditions impact amylase’s activity? Share your results with Kaylie. 1. Does temperature affect amylase activity? If so, at what temperature is amylase most effective? Does this make sense, given where amylase is found in the body? Explain using evidence from your investigation to support your answer.
2. Does pH affect amylase activity? If so, at what pH is amylase most effective? Does this make sense, given where amylase is found in the body? Explain using evidence from your investigation to support your answer.
Enzymes Lab Report Name: ____________________________________ Answer the following questions: 1. Make a table showing the results of your tests for optimal temperature. Based on your results, what is the optimal temperature for salivary amylase? What evidence that you collected supports this conclusion? What was your hypothesis? Did your results support your hypothesis, or if your hypothesis was not supported, did you still learn the optimal temperature range? (4 points)
2. Make a table showing the results of your tests for optimal pH. Based on your results, what is the optimal pH for salivary amylase? What evidence that you collected supports this conclusion? What was your hypothesis? Did your results support your hypothesis, or if your hypothesis was not supported, did you still learn the optimal pH range? (4 points)
3. When conducting laboratory experiments, it is useful to include positive and negative controls to verify that your experimental results are valid. What would be used for positive and negative controls for the Benedict’s assay? For the iodine test? (2 points)
