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Enzyme Activity of Salivary

OBJECTIVES

• Study the action of an enzyme at different pH values and • Compare the profiles of a variety of biological

INTRODUCTION

Perhaps you have noticed that starchy have a faintly sweet in your mouth. You may also know that complex and are related molecules. The chemical structures of all carbohydrates, in fact, are related. For example, is a consisting of many units connected together. The (from plants) and (from animals) are just very large molecules made by connecting glucose rings in different ways. The starch (amylose) shown below contains α−1,4 glycosidic bonds, which are hydrolyzed during by the action of enzymes in and in the .

OH H Digestion () 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 H OH H2O (a ) 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 molecules that catalyze chemical reactions in . 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 . Enzymes use their structures to do several things: attract and bind to the 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 affect the ability of amylase to hydrolyze starch.

We will detect the presence of starch in using 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/ 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 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 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 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 To get a flavor for the differences between various simple carbohydrates, we will taste grade samples of several of these compounds: , , and glucose. Recall that lactose ( ) and sucrose (cane/table sugar) are , 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. We will taste glycine, , , and glutamic .

Since these substances are normally a part of the diet, they are generally safe to taste, but if you have concerns or dietary restrictions about tasting these substances in pure form, participation in this part of the experiment is optional. You may rely on observing your classmates’ reactions and observations rather than doing the tasting yourself.

Enzyme Activity of Salivary Amylase Page 4 Name: ______Pre-lab Questions: Enzyme Activity of Salivary Amylase

1. What is the relationship between starch, dextrins and glucose?

2. What is the job of an enzyme?

3. Draw an diagram that shows the energy in a with and without an enzyme.

4. Cellulose is the rigid material that makes up walls and gives a plant its strength. It is also a polysaccharide made of glucose. What is the major difference between amylose (from starch) and cellulose?

Enzyme Activity of Salivary Amylase Page 5 Name: ______Lab Report Sheet: Enzyme Activity of Salivary Amylase

Part I. Testing enzyme activity in your diluted saliva (pH 7; 37 °C). mL of diluted saliva used: mL of diluted saliva used: Time Observed Starch/Dextrins/Glu Time Observed Starch/Dextrins/Glu (min) Color (Interpretation) (min) Color (Interpretation) 0 0 1 1 2 2 3 3 4 4 5 5 6 6 7 7

Once you have decided on a volume of saliva to use, verify it with your instructor, and make sure to use the same volume for all the remaining parts.

1. What is the best volume of diluted saliva to use? ______

2. How many minutes did it take for the starch digestion to begin? ______

3. How many minutes did it take for the starch digestion to be complete? ______

Part II. Enzyme activity and pH (37 °C) Record the color of the iodine indicator at each pH value and time. Time pH 7 pH 5 pH 6 pH 8 (min) (from part I) 0 1 2 3 4 5 6 7

4. Based on your data, what is the optimal pH (5, 6, 7 or 8) for amylase to hydrolyze starch? Explain your finding.

Enzyme Activity of Salivary Amylase Page 6 Part III. Enzyme activity and temperature

Optimal pH (from part II): ______Record the color of the iodine indicator at each time and temperature. Time 0 C 20 C 37 C 50 C (min) ° ° ° ° 0 1 2 3 4 5 6 7

5. Based on your data, what is the optimal temperature (0, 20, 37 or 50 °C) for amylase to hydrolyze starch? Explain your finding.

6. Look up the typical pH range of saliva. How do your results for optimal salivary amylase activity compare to the pH and temperature conditions expected in your mouth?

7. What can you conclude about the sensitivity of amylase activity to varying conditions in the solution?

Enzyme Activity of Salivary Amylase Page 7 Part IV. Taste tests of biological molecules Substance Type of sugar Flavor notes lactose sucrose fructose glucose 7. All of these sugar molecules have the same nutritional value of about 4 Calories/g, and each is eventually converted to glucose during . What is the possible significance of their different flavor profiles?

Substance Type of R group Flavor notes glycine proline arginine glutamine 8. Which of the R groups above would change form in acidic or basic solutions? Does this give insight into why enzymes are sensitive to changes in pH?

9. Use your textbook or another reference to find and draw the structures of three of the amino acids you tasted. Label each one with its name.

Enzyme Activity of Salivary Amylase Page 8