The Huntington Library, Art Collections, and Botanical Gardens. How Sweet It Is: Enzyme Action in Seed Germination

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1 The Huntington Library, Art Collections, and Botanical Gardens How Sweet It Is: Enzyme Action in Seed Germination Overview This experiment is intended to familiarize students with the macromolecule starch, the action of enzymes (alpha-amylase), and the energy utilization in germinating seeds. Introduction Macromolecules are large molecules made up of small, relatively simple subunits that are strung together. During the process of anabolism, macromolecules are made. Catabolism is the process of breaking them down. Most of the chemical reactions that must occur to either make or break down a macromolecule do not happen spontaneously under normal conditions. For example, starch is a macromolecule made up of many glucose subunits (monomers) joined together. Even when left out in the oxidizing atmosphere, starch will not break down to glucose. Enzymes are the biological catalysts that allow the chemical reactions necessary for metabolism to occur. The seed is a small and hard structure that enables seed plants to survive in adverse conditions. Each seed contains an embryo and stored food. During seed germination, the embryo will use its stored food to grow into a mature plant. In most seeds, the food is stored in the cotyledons, or seed leaves, as starch. Although starch is a storage form of glucose, it cannot pass through living membranes due to the large molecular structure, so before it can be used for energy it needs to be broken down to its constituent sugars. The catabolism of starch is done by alpha-amylase, an enzyme released by the embryo in the seed germination. With the aid of an alpha-amylase, starch is broken down to produce a mixture of one-, two-, or three-monomer-long molecules (glucose, maltose and maltriose). All of the above simple sugars can give a positive test with the Benedict s solution. Benedict s test is used to determine the presence of simple sugars, such as glucose. When Benedict s solution is heated in the presence of low to moderate levels of sugar, the solution turns green; solutions with high glucose levels turn solution yellow or bright orange. Like plants, we need energy too. In our saliva, we have alpha- amylase to breakdown starch in the food we eat. The presence of starch can be detected with iodine. In the iodine test, the starch molecule reacts strongly with iodine to form a deep blue-black complex. If starch is not present no blue-black complex will form. Both Benedict s solution and Iodine solutions are used as indicators. Scientists make use of indicators that give a visual way to see the presence or absence of a compound directly, where normally this compound is undetectable.

2 Motivation The students have been hired by the government as advisors to the Council on Nutrition. A company has introduced into the market a bean sprout that they are advertising as a High Energy bean. To substantiate their claim, the students have been sent to their factory to test these beans and sprouts for sugar and starch. Objectives Upon completion of the lab, students should be able to 1. Compare the molecular structure of common monomers and macromolecules, like glucose and starch. 2. List enzymes used in the breakdown of starch. 3. Define, in the context of chemical reactions, indicator and enzyme. 4. Explain why germinating seeds test positive for sugar rather than starch. Materials Mung bean seeds Sieve or a slatted plant growing flat or moist newspapers for germinating seeds Mortar and pestles (one per group if possible) 15 test tubes for each group Droppers Test tube rack Test tube holder 400-ml beaker Hot plate (or water bath if available) Distilled water (can use spring water) Iodine solution (tincture of iodine works well) Benedict s solution (can buy ready made from Carolina Biological Supply) Associated California Biology Standards 1b. Students know enzymes are proteins that catalyze biochemical reactions without altering the reaction equilibrium and the activities of enzymes depend on the temperature, ionic conditions, and the ph of the surroundings. 1h. Students know that most macromolecules (polysaccharides, nucleic acids, proteins, lipids) in cells and organisms are synthesized from a small collection of simple precursors. 9f. Students know the individual functions and sites of secretion of digestive enzymes, (amylases, proteases, nucleases, lipases), stomach acid, and bile salts. Procedure Advance set-up 1. Place some beans in a beaker and cover them with water (don t use too much water). Place the beaker in a dark warm place. The next day, empty out any extra water. Use a sieve and put some paper towels at the bottom of the sieve. Place the soaked beans on the top of the paper towel. Cover the beans with a layer of paper towel on the top. Wet the whole set-up with water and allow the excess water to drain away at the bottom of the sieve. Rinse the seeds three times a day. Leave the seeds there for approximately 3-4 days to germinate. 2. After the seeds in step 1 have germinated, prepare the dry (un-germinated) beans by soaking some bean seeds in a beaker of water in the refrigerator overnight for the following experiment. The refrigerator stops the enzyme from being active while allowing the seeds to soften so that they may be crushed with mortar and pestle.

3 Part I 1. Fill a 400-ml beaker to about 300 ml with water and heat on the hot plate. 2. Have students in small groups take 5 dry (un-germinated) beans remove the seed coat and crush them in the mortar. To the crushed beans students add 6 ml of distilled water to the mortar and transfer the crushed bean extract to an appropriately marked test tube. 3. After cleaning the mortar and pestle, have students crush the same number of germinated beans and crush this in mortar and pestle. 4. Again add 6 ml of distilled water to the mortar and transfer the crushed germinated bean extract into another appropriately marked test tube. REMEMBER: If a mortar or a dropper is used to transfer the bean extract, it must be washed before every use so you won t contaminate the solutions. 5. Repeat steps 3 and 4 having students crush 5 sets of cotyledons from the germinated seeds into a new test tube. 6. Repeat steps 3 and 4 having students crush 5 hypocotyl and radicle structures (root and lower stem structures) structure from the germinated seeds into a new test tube. 7. Have students add 6 mls of water to a tube as a negative control. 8. Have students let their four tubes sit undisturbed for at least 5 minutes, so that the crushed seed debris can settle to the bottom of the test tubes. Then, have students treat each of the bean extracts as described below. Part II Testing for starch 1. Have students transfer 1 ml of each of their bean extracts or water to separately labeled test tubes. (Be careful not to transfer any debris.) Two drops of iodine solution are then added into each of the 1 ml aliquots. Have students mix and record the color changes on their table. Part III Testing for simple sugars 1. In separate tubes, have students add 2 ml of the bean extract or water from each of their samples (again be careful not to transfer debris) and 6 drops of Benedict s solution into the tube. The tubes should be mixed thoroughly. 2. When the water in the 400-ml beaker boils, students should place their test tubes with Benedict s solution into the water bath and leave in water bath for 10 minutes. REMEMBER: Do not let the water bath boil hard. Control the boiling by turning the hot plate on and off as needed. 3. Students should carefully remove their test tubes with tongs and place the tubes in a test tube rack. Unplug the hot plate to cool. When the tubes cool, students should record the color of the precipitate in each of their tubes. A yellow or orange precipitate will form if large amounts of glucose are present. A green precipitate means no, or very small amounts, of sugar are present. The intermediate between these will appear brown. Evaluation The following questions are listed under the Analysis section of the student handout and may be used as part of a report, class discussion or assessment. 1. Draw a picture of a germinated bean and label cotyledons, hypocotyls and radicle. 2. Draw a picture of the chemical structure of glucose and a starch molecule. Label the structure which is the monomer and which is the polymer. 3. What can you infer about germination and amylase production based on your test results? Where else is the enzyme amylase found? 4. Why are the ungerminated seeds refrigerated overnight? 5. Compare and contrast enzyme utilization in the seed to our own digestion process. 6. Give a hypothesis on why seeds evolved to carrying their food on them. And why do they store that food as starch?

4 7. Identify and explain the controls in this experiment. 8. Explain what an indicator is in science and how it is used. 9. As an official government advisor to the Council on Nutrition, what is your recommendation on the company s truthfulness on marketing the bean sprouts as a high energy food source? What evidence do you offer for that substantiation? Extension Activities 1. This lab may be repeated using bread. Take two small pieces of bread (one small bite size) and add to two separate test tubes and a couple mls of distilled water and let dissolve. To the first test tube add a couple drops of iodine to test for starch. To the second tube add a little spit from students mouths and let sit for 5 minutes. After saliva has time to sit with bread repeat benedict s solution test. This helps students see that both plants and animals have the same enzyme, which performs the same function. 2. Have your students make a model of starch using whatever components they want. 3. Have students use library and Internet sources to write a report on how food crops have evolved to all germinate at the same time. Discuss the science, politics and economics of this evolution. Test Preparation 1. What is the substrate of salivary amylase? (A) protein (B) starch (C) amino acids (D) glucose (E) vitamins 2. Which one of the following would not normally diffuse through the lipid bilayer of a plasma membrane? (A) CO 2 (B) amino acids (C) starch (D) water (E) O 2 3. All of the following are correct about enzyme-catalyzed reactions EXCEPT (A) phosphofructokinase is inhibited by ATP (B) enzymes speed up reactions by supplying necessary energy (C) enzymes lower the energy of activation (D) enzymes often require the presence of minerals (E) all enzymes are not active at the same ph 4. An enzyme is a (A) starch (B) DNA (C) protein (D) lipid (E) chemical

5 Student Sheet: How Sweet It Is; Enzyme Action in Seed Germination Name: Procedure Part I 1. Obtain 5 dry (un-germinated) beans and remove the seed coat and crush them in the mortar. To the crushed beans add 6 ml of distilled water to the mortar and transfer the crushed bean extract to an appropriately marked test tube. 2. After cleaning the mortar and pestle, crush the same number of germinated beans in mortar and pestle. Again add 6 ml of distilled water to the mortar and transfer the crushed germinated bean extract into another appropriately marked test tube. REMEMBER: If a mortar or a dropper is used to transfer the bean extract, it must be washed before every use so you won t contaminate the solutions. 3. Repeat step 3, but crush 5 sets of cotyledons from the germinated seeds into a new test tube. 4. Repeat step 3 with 5 hypocotyl and radicle (root and lower stem structures) structures from the germinated seeds into a new test tube. 5. Add 6 ml of distilled water to a test tube. 6. Let the four tubes sit undisturbed for at least 5 minutes so that the crushed seed debris can settle to the bottom of the test tubes. Part II Testing for starch 1. Transfer 1 ml of each of the bean extracts or water to separately labeled test tubes. Be careful not to transfer any debris. Add two drops of iodine solution to each of the 1 ml aliquots. Mix gently and record the color changes on the table below. Part III Testing for simple sugars 1. In separate tubes, add 2 ml of the bean extract or water from each of the samples (again be careful not to transfer debris) and 6 drops of Benedict s solution into the tube. The tubes should be mixed thoroughly but gently. 2. When the water in the 400-ml beaker (from step 1 in part 1) boils, place the test tubes with Benedict s solution into the water bath and leave in water bath for 10 minutes. REMEMBER: Do not let the water bath boil hard. Control the boiling by turning the hot plate on and off as needed. 3. Carefully remove the test tubes with tongs and place the tubes in a test tube rack. Unplug the hot plate to cool. After the tubes cool, record the color of the precipitate in each of the tubes. A yellow or orange precipitate will form if large amounts of glucose are present. A green precipitate means no--or very small amounts--sugar is present. The intermediate between these will appear brown.

6 Table 1: Iodine test for starch Color Change Interpretation Ungerminated Germinated radicle and hypocotyl Germinated cotyledons Germinated whole sprout Water Table 2: Benedict s test for simple sugars Color Change Interpretation Ungerminated Germinated radicle and hypocotyls Germinated cotyledons Germinated whole sprout Water Analysis On a separate sheet of paper please complete the following: 1. Draw a picture of a germinated bean and label cotyledons, hypocotyls and radicle. 2. Draw a picture of the chemical structure of glucose and a starch molecule. Label which is the monomer and which is the polymer. 3. What can you infer about germination and amylase production based on your test results? Where else is the enzyme amylase found? 4. Why are the un-germinated seeds refrigerated overnight? 5. Compare and contrast enzyme utilization in the seed to our own digestion process. 6. Give a hypothesis on why seeds evolved to carrying their food on them. Why do they store that food as starch? 7. Identify and explain the controls in this experiment. 8. Explain what an indicator is in science and how it is used. 9. As an official government advisor to the Council on Nutrition, what is your recommendation on a company marketing bean sprouts as a high energy food source? What evidence do you offer for that substantiation?

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