Goals: In this lab, students will study different aspects of enzyme activity by doing the following:

A. Establish a method to measure the amount of maltose produced.
B. Determine the effect of different amounts of enzyme on the rate of the reaction.
C. Determine the effect of different amounts of substrate on the rate of the reaction.
D. Determine the effect of pH and temperature on the rate of the reaction.

Students will also learn qualitative and quantitative techniques by using UV-Vis spectrophotometer, vortex mixers and pipet pump dispensers. Students will be able to plot and interpret data obtained from this laboratory exercise and come to conclusions about the activity of salivary amylase.

Students' entering competencies: Before doing this lab, students should understand:

• Safety
• The Nature of Carbohydrates
• Enzymes and Enzyme Activity
• Factors that affect the activity of enzymes: temperature, concentration, time
• Basic principles of spectrophotometry
• Liquid metric measurement and the correct tools for measurement: pipets, Brinkman pipet pump dispensers
• The use of vortex mixers and the importance of mixing soluions in this lab.
• Graphing

1. Industry:                                            Beer/Wine/food
2. Medicine                                           Bacteriology/Pharmaceutical
3. Water Quality
4. Food and Drug Administration)         To analyze food and drugs

Safety:

1. Avoid getting chemicals on hands. Wash hands immediately.
2. Do not drop glassware.
3. CAUTION: The water bath contains hot water. Steam will burn.
4. Be careful when using test tubes in hot water bath. Use a test tube holder.

Lab Techniques:

1. Label all test tubes carefully. Be sure you can identify your set of test tubes. (Class period, group    name or number, test tube number: A1, A2, A3, A4, etc.)
2. Avoid contamination.
3. Hold cuvette on the ribbed part to avoid getting fingerprints on clear surface.
4. Use Kimwipes only to clean fingerprints off clear surface of cuvette.
5. When vortexing, hold test tube at the tip and tilt it away from you.
6. Be careful to add all solutions to test tubes. Check off as you go along.
7. Use one cuvette only when using the spectrophotometer.
8. Be sure to fill the cuvette at least 3/4 full.
9. After using spectrophotometer, pour liquid back into the original test tube.
10. Rinse cuvettes with deionized water before reusing.
11. Before reusing the cuvette, tap excess water out of it. Do not use paper towels, kimwipes, or any other material to wipe cuvette.
12. When placing cuvette in the spectrophotometer, make sure the ribbed side faces the front.
13. Remember timing is critical.
14. Emphasize to students the need to cool test tubes to room temperature.

1. Demonstrate technique of using Spectrophotometer with a Spec-20.
2. Use a flashlight or projector to shine light through a liquid with different concentrations of food coloring to demonstrate absorption.
3. Test a cracker with no salt (or Matzoh). Chew and hold for about two minutes. Check for starch conversion to sugar by taste.
4. Sugar + Benedict's solution in test tube =  no color change
    Matzoh + Benedict's in test tube + boiling = no color change
    Chewed matzoh + Benedict's + boiling = color change to orange
5. Extra credit: students bring in labels/containers of various foods with sugars.
6. Demonstration:
                Cooked liver + H₂O₂ = little reaction, little O₂
                Raw liver + H₂O₂  = more reaction, more O₂
7. Plotting a graph activity.

1. a) There are many macromolecules whose names end in the suffix "-ose." These organic molecules are called:

        ____________________

b) There are many macromolecules whose names end in the suffix "-ase." These organic molecules are called:

        ____________________

2. Name three factors that affect the rate of enzyme activity.

        a)___________________________________

        b)___________________________________

        c)___________________________________

3. a) The water bath in this lab procedure is set for what  temperature? ____________(units)

    b) This temperature is:     cool to the touch           lukewarm             HOT

4. Convert the following:

a) 1000 µL= ______________mL

b) _____________ µL= 20 mL

c) _____________ µL= 1.0 mL

Place the absorbance on the Y axis and the amount of fructose on the X axis.  Draw a smooth curve through the points.  Be sure to include your units.

    ________________________

7. When placing the cuvette into the spectrophotometer for analysis:

a) the sample must fill at least ____________ of the cuvette;

b) the light must pass through the ____________ side of the cuvette.

    chemical is: ________________________
 

9. Use your textbook to look up examples of the following:
 

10. Fill in the following table upon completion of the spectrophotometer analysis. Each group will place their data on the board when they are done.
 
 

Name_________________________________
 
 

 1. In the reaction: H-R-OH + H₂O₂  ¾® R-O + 2 H₂O if the enzyme peroxidase is involved:

a. What are the substrates?
 
 

b. What are the products?
 

2. The colorless molecule guaiacol reacts with hydrogen peroxide (H₂O₂) to form a colored compound.  What is the purpose of this reaction in our experiment and how is the spectrophotometer involved?
 
 
 
 
 
 
 
 
 

3. What do you think will happen to the rate of reaction in question #1 if the amount of enzyme is increased?
 
 
 
 
 
 
 
 

4. What do you think will happen to the rate of reaction in question #1 if the amount of substrate is increased?
 
 
 
 
 
 
 
 

5. What do you think will happen to the rate of reaction in question #1 if the temperature of the reaction is increased to l00^oC?
 
 
 
 
 
 
 
 

You have just finished a lab activity in which you experimented with various factors that affect the activity of salivary amylase. Evaluate the following experiment and see if you can draw a conclusion based on the background and data provided. This will provide you with an opportunity to see if you can apply the principles learned in the lab to a new situation.

How do digestive enzymes function in Paramecia?

Paramecia ingest food particles and enclose them within food vacuoles. Each food vacuole circulates in the cell as the food is digested by enzymes that are added to the vacuole. Nutrients made available during digestion are absorbed into the cytoplasm.

Analysis

1. Some digestive enzymes function best at higher pH levels, while others function best at lower (more acid) pH levels.

2. Congo red is a pH indicator dye; it is red when the pH is above 5 and blue when the pH is below 3 (very acid).

3. Yeast cells that contain Congo red can be produced by adding dye to solution in which the cells are growing.

4. When paramecia feed on dyed yeast cells, the yeast is visible inside food vacuoles.

5. Examine the drawing below. The appearance of a yeast-filled food vacuole "over time" is indicated by the colored circles (labeled due to black and white drawing) inside the paramecium. Each arrow indicates movement and that time has passed.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Critical thinking
 

Analyze what happens to the pH in the food vacuole over time. Explain your conclusions about the sequence of different digestive enzymes that function in paramecium digestion.

Student directions:

You have just finished a lab activity in which you experimented with various factors that affect the activity of salivary amylase. Evaluate the following experiment and see if you can draw a conclusion based on the background and data provided. This will provide you with an opportunity to see if you can apply the principles learned in the lab to a new situation.
 

How does dilution affect enzyme action?

Starch digestion begins in the mouth with enzymes that are secreted in saliva. You decide to see what effect diluting the saliva will have on the digestion of soda crackers, which are mostly baked flour.
 

Analysis

To verify that crackers contain starch, you grind up a cracker and add 5 mL of plain water. You add a drop of iodine solution, and it produces a dark blue color, indicating the presence of starch. Next, you repeat the test but use 5 mL of saliva instead of water. This time, the iodine produces a grayish-purple color instead of the blue color of a positive starch. Apparently, your saliva enzymes had broken down all the starch in the time it took to make the mixture and test it.

Now you decide to see what effect dilution of saliva will have on the digestion of the starch. You place one drop of saliva into 5 mL of water in a test tube, and add the same amount of ground cracker as before. You test the tube at various time intervals and obtain the data shown in the following table:
 
 

Draw a conclusion about the effect of dilution of the enzyme found in saliva on the digestion of starch, and explain the results.

1. What is an enzyme? What is the function of an enzyme?
 
 
 
 

2. Describe the role of each of the following compounds in the Amylase lab:

 A. DNS reagent

B. Amylopectin

C. Amylase

D. Maltose

E. Distilled Water

4. Looking at the lab, do test tubes A1, Bl, and Cl have anything in common? If so, what? Why is this similarity necessary in the lab?
 
 
 
 

5. What would the contents of a test tube be if it had an absorbance of 0.00?
 
 

6. Looking at the flowchart below, what would happen if you eliminated step two of part B or part C?
 

7. Why did you use the hot water bath?
 
 
 
 

8. What would happen if you left the lid off the hot water bath during the five minute time period?
 
 
 
 
 
 

9. The relationship between substrates, enzymes, and products can be represented by the following equation:
 

In a complete sentence, write this equation using the substrate, enzyme, and product in the amylase lab.
 
 
 
 

10. If the optimum temperature for human chemical activity is body temperature (36.6°C), what would you hypothesize would happen to the rate of reaction of amylase as it approached 0°C? What would you hypothesize would happen as the temperature approached 40°C? Explain.
 
 

Use Graph A to answer questions 11-15.

11. What is the substrate? What is the product?
 
 
 
 

12. Does the amount of substrate depend upon the amount of product, or does the amount of product depend upon the amount of substrate? Explain your answer.
 
 
 
 
 
 

13. What is the relationship between the amylopectin and the maltose?
 

14. Where did the information on the volume of maltose come from?
 
 
 

15. According to Graph A, what would happen if you continued to increase the volume of amylopectin? Why? What does this have to do with "maximum rate"?

Use Graph B to answer questions 16-18.

16. Which line is more reasonable? Why?
 
 
 
 

17. What is the relationship between Line A and volume of maltose?
 
 
 
 

18. What does line B indicate?
 
 

 
____________vortex
 
____________micropipettor
 
____________Brinkman pump
 
____________cuvette
 
____________loading UV-Vis spectrophotometer & scanning sample
 
 

 Lactase is an enzyme that works on lactose, a sugar. Lactose sugar is a disaccharide, two rings big. To digest it, you need to break it down into two pieces, two monosaccharides. Lactase enzyme allows you to do this. It reduces the amount of energy needed for the hydrolysis of the disaccharide into two monosaccharides.

Enzymes are proteins. Temperature can effect them. If you cook egg white protein, it changes from clear and runny to firm and white. It is denatured. Enzymes must fit with their substrate in order to work. If an enzyme is denatured, it cannot fit anymore. An enzyme that cannot fit, cannot do its job.

We will test the effect of temperature on how well lactase enzyme works.

First, we must have a way to test whether the enzyme is working or not. We will use an indicator that changes color. TesTape is the indicator. It changes color in the presence of glucose, a monosaccharide.

IF THE ENZYME IS WORKING, IT WILL PRODUCE GLUCOSE.

IF THE ENZYME IS NOT WORKING, IT WILL NOT PRODUCE GLUCOSE.
 

We will see how well the enzyme works after being subjected to different treatments.

We will test:

1. milk + enzyme (our control)

2. milk + boiled enzyme (Does boiling stop the enzyme from working?)

3. boiled milk + enzyme (Does boiling the substrate stop the enzyme from working?

Hint: is the substrate a protein or a sugar?)

4. plain, untreated milk (Does milk have glucose without enzyme treatment?)

All the milk we are testing has had enzyme added to it already, because the enzyme works too slowly for us to add it in class.

To test the various combinations, make a serial dilution of the enzyme-treated milk: pure, 1:10, 1:100. Do this for each of the 4 kinds of combinations. Test each with TesTape. Repeat for 3 trials of each combination.
 

Most people make lactase enzyme when they are young. As they get older, milk is not as important a part of their diet. Many people no longer make lactase as they get older. They then have trouble digesting dairy products. The lactase enzyme they can no longer produce for themselves can be added to dairy products before they eat them. That is why it was easy to buy lactase for the experiment. Do not try adding lactase to food without following package directions and without parental supervision.

 Extend your lactase lab to investigate the effects of pH on lactase action. Most enzymes can only do their work within a relatively narrow range of pH values. Should the environment become too acidic or too alkaline (too basic), the enzyme becomes denatured and cannot work. Different enzymes work best under different conditions. One that must work in the acidic environment of the stomach differs in this way from one that operates in the more basic interior of the small intestine.
 
Using a pH meter or universal indicator paper, make a vinegar/distilled water solution of pH 3. Make a sodium bicarbonate /distilled water solution of pH 10.

Test a control solution. In the bottom right well of your culture plate, put 5 drops of distilled water and 1 drop of lactose solution. Test the pH of the mixture. Add one drop of lactase solution. Wait 5 minutes and test for glucose production with TesTape.

Starting from well A-l, make a serial dilution of the vinegar solution from pure to 1:1000. Add a drop of lactose solution to each well. Test and record the pH of each well. Add a drop of lactase solution to each well. After 5 minutes, test each mixture for glucose production with TesTape.

Repeat the vinegar test for 2 more rows. Did you get similar results each time?
 
Make a serial dilution of the sodium bicarbonate/distilled water solution from pure to 1:1000. Add a drop of lactose solution to each well. Test and record the pH of each well. Add a drop of lactase solution to each well. After 5 minutes, test each mixture for glucose production with TesTape.
 
Repeat the sodium bicarbonate solution for 2 more rows.
 
How could you modify this experiment to make it more precise? more accurate? What other acids and bases could make the experiment more realistic? Could any other chemical interactions modify your results in the changed experimental conditions?