LAB 3: DIGESTION OF ORGANIC MACROMOLECULES INTRODUCTION Enzymes are a special class of proteins that lower the activation energy of biological reactions. These biological catalysts change the rate of chemical reactions without being consumed or altered by the reactions. The substance which an enzyme acts on is referred to as the substrate. The substrate (or substrates) binds to a special region on the surface of the enzyme called the active site. As the substrate enters the active site, it induces the enzyme to change its shape slightly. This induced fit results in the formation of an enzyme-substrate complex, which facilitates the conversion of substrate to product. ENZYME + SUBSTRATE(S) ENZYME-SUBSTRATE COMPLEX ENZYME + PRODUCT The activity of an enzyme is affected by general environmental factors (i.e. temperature and ph) and by special molecules which influence the catalytic ability of the enzyme (i.e. inhibitors, activators and cofactors). The action of digestive enzymes is to bring about hydrolysis. Hydrolysis is a chemical process whereby water is added across a covalent bond resulting in splitting or lysis of the molecule. Enzymatic digestion involves the hydrolysis of organic macromolecules such as proteins, carbohydrates and lipids. PURPOSE: 1. to monitor the activity of digestive enzymes 2. to evaluate the substrates and products of the reactions catalyzed by digestive enzyme 3. to review the functions of various digestive enzymes 4. to evaluate factors that affect the activity of various digestive enzymes 3-1
REVIEW: Before beginning this lab, complete the table that follows. Indicate where in the body the cells are located that secrete each enzyme (be as specific as possible). Indicate the substrate and the products in the reaction catalyzed by each enzyme. ENZYME SECRETED BY SUBSTRATE PRODUCT(S) salivary amylase pancreatic amylase disaccharidases (maltase, sucrase, lactase) lipase pepsin chymotrypsin trypsin (exo)peptidases PART I: DIGESTION OF STARCH BY SALIVARY AMYLASE Enzymatic digestion begins in the mouth. Enzymes tend to be very substrate specific in the reactions they catalyze. An enzyme can distinguish its substrate from even closely related compounds. This specificity is based on the shape of the active site, and ensures that each enzyme catalyzes only one type of chemical reaction. Salivary amylase will be used to investigate substrate specificity. This enzyme is secreted into the mouth as a component of saliva. Amylase hydrolyzes the covalent bonds between glucose molecules in certain polysaccharides. Polysaccharides differ in the amount of branching and the type of covalent bond between the glucose molecules. Not all plant polysaccharides however, can be digested. Undigestible polysaccharides are excreted in the feces, and comprise the fiber component of our diet. Benedict s reagent** will be used to monitor the products of this reaction. Benedicts reagent produces a coloured precipitate in the presence of all monosaccharides and some disaccharides, following heating. blue orange/yellow ppt no sugars present sugar present ** CAUTION! Benedict s reagent is a very strong base. Avoid contact with the skin. Dispose of Benedict s solutions in special waste container provided. 3-2
Procedure Prepare 6 s as outlined below. contents 1 2.0 ml glucose 2 2.0 ml maltose 3 2.0 ml starch 4 2.0 ml cellulose 5 1.5 ml 1% cellulose + 0.5 ml saliva 6 1.5 ml 1% starch + 0.5 ml saliva Mix well, then incubate all 6 tubes at 37ºC for 30 minutes, Test the contents of each for the presence of sugars by adding 20 drops of Benedict s reagent and heating each tube in a boiling water bath for 5 minutes. Record your observations in Table 1. Table 1. Substrate specificity of salivary amylase Test contents tube 1 1% glucose 2 1% maltose 3 1% starch 4 1% cellulose 5 1% cellulose + 0.5 ml saliva 6 1% starch + 0.5 ml saliva Benedict test colour conclusions QUESTIONS: 1. Which of the polysaccharides tested does salivary amylase hydrolyze? 2. What is the product of starch hydrolysis by amylase? 3-3
PART 2. EFFECT OF ph ON HYDROLYSIS OF PROTEINS BY PEPSIN Each enzyme has a ph optimum at which it is most able to bind substrate. The ph optimum for most enzymes is between ph 6 and ph 8, but there are many exceptions. The enzyme pepsin will be used to investigate ph optima. Biuret reagent** will be used to test for the presence of proteins and peptides. Biuret reagent reacts with the peptide bonds in proteins and peptides to yield solutions that differ in colour, as follows. Record the results in Table provided. blue purple pink no proteins or peptides proteins present peptides present ** CAUTION! Biuret reagent is a very strong base. Avoid contact with the skin. Dispose of Biuret solutions in special waste container provided Procedure Prepare 5 s as follows. Mix the contents well. Using ph test strips, record the initial ph of each solution in Table 2, then allow each tube to incubate at 37ºC for 45 min. contents 1 6 ml water 2 4 ml water + 2 ml 1% albumin 3 2 ml water + 2 ml 1% albumin + 2 ml 1% pepsin 4 2 ml 1.0% HCl + 2 ml 1% albumin + 2 ml 1% pepsin 5 2 ml 1.0% NaOH + 2 ml 1% albumin + 2 ml 1% pepsin At the end of the incubation period, test each tube for the presence of proteins / peptides by the addition of 20 drops Biuret reagent. Record results in Table 2. Table 2 : Effect of ph on hydrolysis of proteins by pepsin ph Biuret test colour conclusions 1 2 3 4 5 3-4
QUESTIONS: 1. What ph (acidic, basic or neutral) appears to be optimal for the enzyme pepsin? 2. Into what compartment of the gastrointestinal tract is pepsin secreted? 3. What is the ph of this compartment? 4. What is secreted into this compartment that affects the ph? PART 3: EFFECT OF BILE ON HYDROLYSIS OF LIPIDS During the digestion of lipids, lipase and bile salts are released into the upper part of the small intestine (the duodenum). Bile is an emulsifier produced in the liver from cholesterol, and stored in the gallbladder. Emulsifiers (like detergents and soaps) help to disperse water insoluble lipids in aqueous solutions. This emulsification in turn, facilitates hydrolysis of the ester bonds in triglycerides by pancreatic lipase. The rate of this reaction can be monitored by measuring ph. Hydrolysis of fats releases fatty acid molecules, which lower the ph of a solution. The ph indicator phenolphthalein is colourless in neutral or acidic conditions, and pink in the presence of a base. As fatty acids are produced a solution containing phenophthalein will change from pink to colourless. The more quickly this colour change occurs, the faster the rate of lipid hydrolysis. Procedure Prepare 3 s as follows. contents 1 1 ml cream + 4 ml water 2 1 ml cream + 1 ml water + 3 ml 0.1% lipase 3 1 ml cream + 1 ml bile solution + 3 ml 0.1% lipase Before incubating the tubes, add 2 drops of phenolphthalein. Then add enough NaOH (1 drop at a time, mixing well after each drop) until tube contents turn a persistent pink colour. Mix well by inverting the tubes, then incubate on the bench in a beaker of warm water. Monitor the tubes at 3 minute intervals. Record the time required for the tubes to change from pink to colourless in Table 3. Table 3 : Effect of bile on hydrolysis of lipids by pancreatic lipase initial colour colour after incubation (minutes) 1 Time for colour change (min) 2 3 3-5
QUESTIONS: 1. Most of the fats in foods are in the form of triglycerides. Draw the structure of a triglyceride, and label the glycerol and fatty acid components. 2. In which of the tubes were fatty acids released most rapidly from the triglycerides? Explain these results: 3. What ph do you think would be optimal for pancreatic lipase? (THINK! Where does lipid digestion occur, and what is the ph of this digestive compartment?) 4. What is a cholecystectomy and how might this affect an individual s ability to digest dietary fat? 3-6