LAB 3: Biomolecules and Digestion

Page 3.1 LAB 3: Biomolecules and Digestion Food taken into our bodies must first be broken down by mechanical and chemical digestion before it can be absorbed and used as an energy source. The chemical digestion is completed by specialized enzymes. In this lab, you will demonstrate the enzyme-catalyzed hydrolysis of proteins, lipids, and carbohydrates. You will test for the presence of the original substrate (protein or carbohydrate) and for the presence of its hydrolysis products (amino acids or monosaccharides). These experiments will help you understand the 4 types of large biomolecules and their structures and functions Objectives At the conclusion of this laboratory the student will understand and be able to describe: 1. enzyme catalyzed hydrolysis and the building blocks of proteins, fats, and carbohydrates. 2. how to design an experimental protocol to test for enzyme-catalyzed hydrolysis, including proper positive and negative controls. 3. the various tests (assays) used to determine if hydrolysis of the proteins, lipids, and carbohydrates has occurred. 4. which enzymes digest the three major nutrients (proteins, lipids, and carbohydrates) and where these enzymes are produced. 5. what factors affect the activity of enzymes. 6. expected results when hypothetical situations similar to the experiments performed are presented. VOCABULARY LIST Table 1: Use this list to prepare for quizzes and lab practicals. digestion chemical digestion mechanical digestion intracellular metabolism hydrolytic enzymes or hydrolases substrate product pepsin peptide chyme dipeptidases and aminopeptidases bile salts micelles lipase chylomicrons lacteals enzymecatalyzed hydrolysis trypsin, chymotrypsin, and carboxypeptidase albumin biuret test ninhydrin test emulsification ph indicator positive control negative control starch amylase disaccharides monosaccharides maltase iodine test Benedict s test carbohydrates proteins lipids

Page 3.2 This lab will consist of 2 activities: ACTIVITY 1. Protein Digestion and the Effect of Denaturation on Enzyme Activity Students will perform protein digestion, detect the products of protein hydrolysis, and determine the effect of enzyme denaturation on protein digestion. ACTIVITY 2. Carbohydrate Digestion and the Effect of ph and Denaturation on Enzyme Activity Students will perform carbohydrate digestion, detect the products of carbohydrate hydrolysis, and determine the effect of ph and denaturation on enzyme activity BACKGROUND AND REFERENCES In this lab, you will demonstrate the enzyme-catalyzed hydrolysis of proteins and carbohydrates. You will test for the presence of the original substrate (protein or carbohydrate) and for the presence of its hydrolysis products (amino acids or monosaccharides). The absorption of nutrients from foods that are consumed first requires that those foods be broken down into small absorbable units by the process of digestion. Digestion in the human body includes both a mechanical phase (chewing, mixing of chyme, peristalsis), and a chemical phase. Chemical digestion occurs with the aid of special proteins known as digestive enzymes. After being synthesized in specialized cells, digestive enzymes are released into the digestive tract to accelerate the extracellular breakdown of the biological nutrients (carbohydrates, proteins, and lipids) in the food and fluids consumed. The products of chemical digestion are absorbed into the bloodstream or lymphatic vessels, carried to the cells of the body, transported into these cells, and then used in intracellular metabolism. You are probably familiar with the various types of intracellular metabolism, such as aerobic respiration, glycolysis, lipolysis, protein synthesis, etc. The extracellular digestion of nutrients occurs by a process known as enzyme-catalyzed hydrolysis. Hydrolytic enzymes or hydrolases are used to catalyze or speed up the chemical reaction where water is added to help break down larger food molecules into their monomers. Your textbook reviews the hydrolysis of the 3 major nutrient groups and demonstrates that: Proteins + H20 + enzyme à amino acids + enzyme Fats + H20 + enzyme à fatty acids + glycerol (or monoglyceride) + enzyme Carbohydrates + H20 + enzyme à monosaccharides + enzyme In the body, each of the above reactions can only occur if the proper hydrolytic enzyme is present and active. Notice that the enzymes are not used up or changed in the reaction; they are present on both the reactant (substrate) side and the product side of the equation. The specific enzymes for the digestion of carbohydrates, proteins, and lipids are discussed in the following sections. You may wish to review in your textbook how enzymes act as catalysts and which factors control their activity, such as ph, temperature, enzyme and substrate concentrations, and cofactors.

Page 3.3 Checkpoint: 1. Which of these is absorbed into the bloodstream in the GI tract: macromolecules, or their digested breakdown products? 2. What would you predict the optimal temperature to be for digestive enzymes that work in the human body? 3. What would you predict the optimal ph to be for pepsin; for the other enzymes? 4. What is the difference between mechanical digestion and chemical digestion? ACTIVITY 1: Protein Digestion Protein digestion begins in the stomach with the enzyme pepsin. Pepsin is produced by the chief cells of the stomach and hydrolyzes proteins into smaller peptides (short strands of amino acids). Pepsin works best at a stomach ph of 2 and denatures when the chyme (partially digested food) moves into the small intestine where the ph is around 9. Hydrolysis of proteins into peptides is continued in the small intestine by the pancreatic enzymes trypsin, chymotrypsin, and carboxypeptidase. The peptides produced by these first four enzymes are further broken down into amino acids by aminopeptidases and dipeptidases, which are enzymes that are produced in the brush border of the small intestine. The amino acids are then absorbed into the capillaries of the small intestine and carried to the cells of the body for intracellular metabolism. You will be observing the digestion of protein (albumin) by the enzyme trypsin according to the following hydrolysis reaction: Protein + trypsin + H2O à peptides + trypsin You can monitor the progress of the above reaction using two tests, the biuret test for the presence of protein and the ninhydrin test for the presence of amino acids. Before the reaction takes place, the biuret test should be positive and the ninhydrin test negative. After the reaction has gone to completion (all of the protein has been hydrolyzed to amino acids), the biuret test should be negative and the ninhydrin test should be positive. For the biuret test, the biuret reagent turns from blue to violet when positive for proteins. A pink color in the biuret test indicates partial digestion of the protein into polypeptides. For the ninhydrin test, a color change from colorless to blue-violet is positive for the presence of amino acids. A Note on Lipid Digestion Because lipids are not water soluble, they must first be emulsified before enzymes can efficiently break them down. In emulsification, bile salts break the large lipids into minute oil droplets. Bile salts are produced by the liver, stored in the gallbladder, and released into the small

Page 3.4 intestine when needed. The small oil droplets, called micelles, have more surface area available for the enzymes to act upon. Lipase is an enzyme produced in the pancreas and digests lipids according to the following hydrolysis reaction: Lipid (olive oil) + lipase + H2O à monoglycerides (or glycerol) + fatty acids + lipase If you eat a lipid such as olive oil, the olive oil is the substrate for the enzyme while the glycerol and fatty acids are the products of the hydrolysis reaction. The glycerol and fatty acids are absorbed into the epithelial cells of the small intestine and packaged into special lipoproteins known as chylomicrons. The chylomicrons are absorbed into the lacteals (lymphatic vessels) of the small intestine and carried to the liver for intracellular metabolism. Using lipase outside of the body you can usually monitor the progress of the above reaction using a ph indicator. Because the breakdown products of lipids include fatty acids, the ph of a solution in which lipids have been hydrolyzed will decrease. This decrease in ph can be detected with a ph meter or with a ph indicator paper. We will not be performing any experiments with lipase in our lab because the water in our labs is acidic and does not allow for measurement of slight acidic changes. However, your lab instructor may show you an example of emulsification during lab. ACTIVITY 2: Carbohydrate Digestion You will be observing the hydrolysis of carbohydrate (starch) by the enzyme amylase. Salivary amylase is produced in the salivary glands while pancreatic amylase is made in the pancreas. Both digest starch according to the following hydrolysis reaction: Starch + amylase + H2O à maltose + amylase Starch is the substrate for the enzyme amylase while maltose is the product of the hydrolysis reaction. Maltose is a disaccharide which is further broken down into two glucose units (monosaccharides) by the enzyme maltase. Maltase is found in the brush border of the small intestine. The resultant glucose is then absorbed into the capillaries of the small intestine and carried to the cells of the body for intracellular metabolism. You can monitor the progress of the above reaction using two tests, the iodine test for the presence of starch and the Benedict's test for the presence of maltose. Before the reaction takes place, the iodine test should be positive and the Benedict's test negative. After the reaction has gone to completion (all of the starch has been hydrolyzed), the iodine test should be negative and the Benedict's test positive.

Page 3.5 Expected results for the iodine test include: Iodine + starch à blue-black color +++ positive Iodine + partially digested starch à purple-red color ++ partially positive Iodine + almost completely digested starch à brown-red-orange + slightly positive Iodine + completely digested starch à yellow color - negative Iodine + no starch à yellow color - negative Expected results for the Benedict's test include: Benedict's solution + starch à blue color- negative Benedict's solution + partially digested starch à greenish-blue ++ slightly positive Benedict's solution + digested starch à yellow-orange ++ partially positive Benedict's solution + completely digested starch àred color (precipitate) +++ positive Checkpoint: Please fill out Table 2. Experimental protocol tubes to demonstrate enzyme catalyzed hydrolysis This table will allow you to summarize the contents of the tubes used in this exercise. Type of Digestionà Protein Digestion Lipid Digestion Carbohydrate Digestion Contents Substrate (Reactant) Albumin Enzyme Pancreatic lipase Expected Product Assay that will be done for substrate (include name and + vs. result) Assay that will be done for product (include name and + vs. result) Ninhydrin test + = blue-violet color - = colorless Maltose Iodine test + = blue-black color - = brown-yellow color

Page 3.6 EXPERIMENTAL PROCEDURES The experimental procedure for today s lab involves three parts: 1. Set up various conditions under which hydrolysis will occur a. Presence or absence of enzyme b. Presence or absence of substrate c. Active enzyme versus denatured enzyme d. Various ph environments 2. Allow hydrolysis to occur in an environment similar to the body a. Incubate for 30-60 minutes at 37 C 3. Assay the tubes to determine if enzymatic digestion has occurred a. Is the substrate still present or is it absent? b. Is the product (the result of hydrolysis) present? FLOW SHEET: If you do the activities in this order, you will have plenty of time to do all of the tests. Each pair of students will have a group symbol so they can tell their tubes from those of other students. Circle each number below after you have carried out the procedure described. 1. Label your test tubes in the following manner: "group symbol--test letter--tube letter". For example, for the "star" group, the protein test, and tube B, the tube would be labelled "*PB" Protein digestion: (6 tubes) *PA-*PF Carbohydrate digestion: (4 tubes + 3 tubes) *CA-*CD and *CF- *CH Benedict's assay after digestion: (4 tubes) *BA-*BD Write directly on the glass near the top of the tube--not on the label area. Label 4 glass pipettes as A-D and 4 spot plate wells A-D. 2. Set up your protein digestion tubes (step 2 in activity 1). The tubes from step will need to incubate for up to 2 hours, so please note the time. 3. Set up your carbohydrate digestion tubes CA-CD (step 2 in activity 2). The tubes from step 2 will need to incubate for 30 minutes, so please note the time. 4. Do the protein and carbohydrate control assays (step 1 in activity 1; step 1 in activity 2). Also use this time to predict results for all tests if you have not already done so. 5. Perform the carbohydrate digestion tests (tubes CF-CH) (step 4 of activity 2). 6. Analyze your carbohydrate digestion tubes (step 3 in activity 2). 7. Analyze your protein digestion tubes (step 3 in activity 1).

Page 3.7 ACTIVITY 1. Protein Digestion and the Effect of Denaturation on Enzyme Activity Step 1. Perform control biuret and ninhydrin assays. CAUTION: Ninhydrin is toxic. Wear gloves. Biuret assay: Place 3 ml of 1% albumin solution in a test tube and add 3 ml of 10% sodium hydroxide and 3 drops of biuret reagent (1% CuSO4. 5H2O). If the solution does not turn violet, continue adding and counting drops of biuret reagent until the solution turns violet. This will be the number of drops you will add to your experimental tubes. Ninhydrin assay: Place 5 ml of 0.1% amino acid and 0.5 ml of 0.5% ninhydrin in a test tube. Place the tubes in a boiling water bath for 5 minutes. Observe and record the color. The solution should be blue-violet if amino acids are present. Step 2. Determine the effect of ph and denaturation on enzyme activity. Obtain and label 6 test tubes (PA-PF). Place the reagents in the test tubes as directed in Table 3. The enzyme pancreatin, which contains trypsin, should be the last substance added to each tube. The time should be recorded when the enzyme is added for each tube. Each tube must be shaken well upon addition of the enzyme. Using ph strips, record the ph of each tube in table 3. Place the tubes in a 37 o C water bath. Swirl the tubes every 15 minutes to keep the contents mixed. The tubes will need to incubate up to 2 hours. Step 3. Perform a biuret and a ninhydrin assay on the contents of the tubes after 2 hours of incubation. Biuret assay: put 3 ml of solution from each tube (PA-PF) into a new labeled test tube. Add the appropriate number of drops of biuret reagent (determined above in the control test) to each tube. Record your results in Table 3. Ninhydrin assay: put 5 ml of solution from each test tube (PA-PF) into new labeled tubes. Add 0.5 ml of 0.5% ninhydrin (CAUTION) into each tube and place them in a boiling water bath for 5 minutes. Record your results in Table 3.

Page 3.8 Checkpoint: Record the results of Activity 1 in the table below. Table 3. Reagent PA PB PC PD PE PF 1% albumin 5 ml 5 ml 5 ml 5 ml 5 ml 0.1 M HCl 5 ml 0.02 M NaOH 5 ml 5 ml 5 ml 5 ml Water 5 ml 2 ml 5 ml 5% pancreatin (trypsin) 2 ml 2 ml 2 ml 2 ml Boiled 5% pancreatin 2 ml (trypsin) ph of tube Biruet assay results Ninhydrin assay results ACTIVITY 2. Carbohydrate Digestion and the Effect of ph and Denaturation on Enzyme Activity Step 1. Perform control iodine and Benedict's assays. Iodine assay: Place 1 drop of 1% starch solution in a spot plate. Add 1 drop of the iodine solution to the plate. Observe and record the color: Benedict's assay: Place 2 ml of 5% maltose solution and 2 ml of Benedict's reagent in a test tube. Place the test tube in a boiling water bath for 3 minutes maximum. Often, a color change is observed after 45 seconds. Observe and record the color: Step 2. Determine the effect of ph and other factors on enzyme activity. Obtain and label 4 test tubes (CA-CD). Place the reagents in the test tubes as directed in Table 5. The substrate, starch, should be the last substance added to each tube and the time should be recorded when the starch is added to each tube. Each tube must be shaken well upon addition of the starch. Place the tubes in a 37 o C water bath for 30 minutes. Step 3. Perform an iodine assay and a Benedict's assay on the tube contents after 30 minutes of incubation. Iodine assay: place one drop of the carbohydrate reaction tube contents (from tubes CA- CD) into appropriately labeled spot plate wells (A-D). Add 1 drop of iodine solution to each well. Record your results below. The iodine assay is testing for the presence of.

Page 3.9 Benedict's assay: place 5 ml of the carbohydrate reaction tube contents (from tubes CA- CD) into appropriately labeled tubes (BA-BD). Add 2 ml of Benedict's reagent to each tube and boil for 3 minutes maximum. Record your results in the table below. The Benedict s assay is testing for the presence of. Checkpoint: Record the results of Activity 2 in the following tables. Table 4. Table 5. Reagent CA CB CC CD CF CG CH 1% amylase 2 ml 2 ml 2 drops 2 drops 2 drops Boiled 1% amylase 2 ml 100 mm phosphate 2 ml buffer (ph 3) 100 mm phosphate 2 ml 2 ml 2 ml 2 ml 2 ml buffer (ph 7) 100 mm phosphate 2 ml buffer (ph 10) Water 2 ml 5 ml 1% starch 5 ml 5 ml 5 ml 2 ml 2 ml 2 ml Results of iodine Time to + iodine result: assay Results of Benedict s assay Step 4. Obtain and label 3 test tubes (CF, CG, CH). Place the reagents in the test tubes as directed in Table 5 (above). The substrate must be added as the very last reagent and you must be ready to begin timing immediately upon adding the starch. Each tube must be shaken well upon addition of the enzyme. These tubes will be used to determine the effect of ph on enzyme activity. They should be incubated at room temperature and you should perform an iodine test on the contents every 30 seconds. Record the time when a negative iodine test occurs in Table 5 above. Some of the tubes may not produce negative results. If this is the case, stop observing them after 5 minutes.