Enzyme Activity of Salivary Amylase OBJECTIVES • Study the action of an enzyme at different pH values and temperatures • Compare the flavor profiles of a variety of biological molecules INTRODUCTION Perhaps you have noticed that starchy foods have a faintly sweet taste in your mouth. You may also know that complex carbohydrates and sugars are related molecules. The chemical structures of all carbohydrates, in fact, are related. For example, starch is a polysaccharide consisting of many glucose units connected together. The polysaccharides cellulose (from plants) and glycogen (from animals) are just very large molecules made by connecting glucose rings in different ways. The starch (amylose) molecule shown below contains α−1,4 glycosidic bonds, which are hydrolyzed during digestion by the action of enzymes in saliva and in the small intestine. OH H Digestion (hydrolysis) of starch into glucose H O HO HO H H OH H OH STARCH (AMYLOSE) H O n = 300 - 3000 glucose units H O HO H H OH H OH H O H O HO H H OH amylase H OH H2O H O H O n HO H H OH amylase MALTOSE H OH DEXTRINS H2O (a disaccharide) maltase H2O H OH H OH H O OH H H OH H O HO HO HO H H O H O HO H H OH HO HO H OH HO H HO H H OH H OH H OH H OH GLUCOSE H OH H OH Enzyme Activity of Salivary Amylase Page 1 What is not obvious from this picture is that starch is actually a helical molecule, with a shape similar to a telephone cord. When mixed with saliva, these starch molecules are hydrolyzed by the enzyme amylase into shorter sections of helix called dextrins. Eventually, amylase further breaks apart the dextrins to form maltose (a disaccharide), which is finally broken down to glucose by maltase, an enzyme found in the lining of the small intesting. Enzymes are folded protein molecules that catalyze chemical reactions in biochemistry. Your body has hundreds of different types of enzymes that help carry out all the chemical reactions that you need to live. It might be helpful to think about an enzyme like a bridge that makes getting from one place to another easier. Although it is possible to cross a creek, river or bay without a bridge, it is impractical to do so for daily life as we know it. For both bridges and enzymes, structure and shape are very important for proper function. Enzymes use their structures to do several things: attract and bind to the substrate molecule(s), help them to react, and then release them so new molecules can come in. As you can imagine, this is a complex process, and it is sensitive to the conditions of the reaction. In this experiment, we will study how pH and temperature affect the ability of amylase to hydrolyze starch. We will detect the presence of starch in solution using iodine solution as an indicator. Iodine (I2) is a deep blue/black in the presence of starch. As starch is broken up to dextrins, the iodine turns to a brown/red color, followed by a pale brown/yellow when the enzyme has completed hydrolysis. You will use the color changes of iodine to see how far the reaction has progressed at different times. Stage of hydrolysis Color of iodine indicator Starch Deep blue/black Dextrins Dark brown/red Pale brown/yellow Maltose or Glucose (no change) PROCEDURE Part I. Preparing a solution of amylase and initial testing of enzyme activity Salivary amylase is a powerful enzyme, and in order to study it, we will need to dilute it. Begin by collecting 1 mL of saliva in a graduated cylinder. Use your squeeze bottle to wash the saliva into an Erlenmeyer flask, and dilute it to a volume of about 50 mL. Mix thoroughly with a stir plate and stir bar to make sure the enzyme is spread evenly through your diluted saliva. Since everyone’s saliva is a little different, you will need to find out what quantity of your diluted saliva will hydrolyze the added starch in 5-7 minutes. Obtain a 24-well plate, and Enzyme Activity of Salivary Amylase Page 2 label the first two columns with times from 0-7 minutes (setting it on a piece of paper is a good way to do this). You will find conditions that allow your starch digestion to be complete within this time period. Add 4 drops of brown iodine solution to each of the labeled wells. The iodine will indicate the progress of the starch digestion and also stop the enzyme from digesting the starch any further. To test your saliva’s activity, use a disposable plastic pipet to add 1 mL of your diluted saliva solution to a test tube (you will reuse this pipet for the whole experiment). In a second test tube, mix 1 mL (20 drops) of pH 7 buffer and 2 mL of 1% starch. Put both test tubes in a 37 °C water bath for a few minutes to allow them to warm up to physiological temperature. When you are ready, get a clean plastic pipet ready and pour the buffered starch solution into your saliva solution and mix well. Start a stopwatch and take a sample of the mixture right away. Add 5 drops of the reaction mixture to the well labeled “0 minutes,” and squirt any extra mixture back into the reaction (reuse this pipet for the whole trial). Each minute the reaction goes, take another sample of the mixture and add 5 drops to the next well. Keep the reaction in the water bath during this process so that it stays at the expected temperature. At the end of the trial, swirl the well plate to make sure the solutions are mixed well, and decide whether the reaction finished within the 5-7 minute window. If the reaction is not done within this window, set up the next two columns (8 wells) in your plate with iodine solution. Check with your instructor, and then repeat the procedure with a different amount of diluted saliva until you find an amount that results in complete reaction within 5-7 minutes. Once you have worked out how much diluted saliva to use, record your data: the color of each solution, and your interpretation of whether the mixture is still starch, or has turned to dextrins or plain glucose. If you can, take a picture of this run and save it (do not dump it out) so you can compare it to your other trials. Make sure your plate is well- labeled in your picture so you don’t have to guess what you are looking at. Enzyme Activity of Salivary Amylase Page 3 Part II. Enzyme activity and pH Now that you have established a set of conditions that causes hydrolysis of your added starch in 5 to 7 minutes at pH 7, you will test the effect of changing the pH. Set up and label a new 24-well plate for three trials: one each at pH 5, 6 and 8. Each trial will take up two columns of the plate. You will test the enzyme activity as you did at pH 7, still using the volume of diluted saliva you found worked best in Part I. The procedure for these tests is the same as in Part I, but use the buffer of the appropriate pH for each test. Use the water bath to keep the temperature at 37 °C for all these trials. When your runs are completed, take a picture of the labeled well plate and record your results (along with your interpretation of them) in the table provided. What is the optimal pH (5, 6, 7, or 8) for amylase to break down starch? Part III. Enzyme activity and temperature To test the temperature dependence of amylase activity, choose the pH that resulted in the fastest hydrolysis of starch. Set up a third 24-well plate for three trials, this time always at the pH you choose, but with three different temperatures: about 0 °C, 20 °C and 50 °C. The 0 °C trial you will keep in an ice water bath instead of a warm water bath. The 20 °C trial can be placed in a room temperature water bath, and the 50 °C trial will be done in another hot water bath. In each trial, let the two test tubes sit in their baths for a few minutes before mixing them, and take samples at regular time intervals into your well plate. Record your results and interpretations as before, and take a picture of the labeled well plate for comparison. Part IV. Taste testing simple carbohydrates and amino acids To get a flavor for the differences between various simple carbohydrates, we will taste food grade samples of several of these compounds: lactose, sucrose, fructose and glucose. Recall that lactose (milk sugar) and sucrose (cane/table sugar) are disaccharides, while fructose and glucose are monosaccharides. To taste them, obtain a clean tasting stick, and dip it in water, then a sample of the sugar (then taste it). No double dipping! We will do this together as a class. To avoid cross-contamination, we will not use lab glassware for this part of the procedure. Place the used stick in the waste container provided, and record your observations about each sugar’s flavor. We will also taste a selection of pure amino acids sold as nutritional supplements. Use the same procedure to taste these as you did for the sugars.