Pre-Lab For Amylase Lab
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.
Synopsis: This lab will help students learn about the nature of sugars and measurement
of enzymatic activities affected by pH, temperature, and concentration of sugars.
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:
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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
REAL WORLD APPLICATIONS
1. Industry:
2. Medicine
3. Water Quality
4. Food and Drug Administration)
Beer/Wine/food
Bacteriology/Pharmaceutical
To analyze food and drugs
SAFETY AND LAB TECHNIQUES
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.
Suggested Pre-lab Demonstrations and Related Activities or the teacher
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.
FACTORS AFFECTING THE ACTIVITY OF SALIVARY AMYLASE
PRELAB HANDOUT: WORKSHEET
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
5. Practice graphing the following data on to a blank graph.
Amount of fructose (µL) Absorbance
0
0.00009
100
0.18247
300
0.20962
500
0.23454
700
0.29006
1000
0.41083
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.
6. Tubes #A1, B1, and C1 contain no maltose, enzyme, or substrate. The purpose of these
tubes is to serve as the
________________________
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.
8. There is one chemical in this lab that especially deserves caution because it is a skin
irritant. The name of this
chemical is: ________________________
9. Use your textbook to look up examples of the following:
Carbohydrates
Real Name
Common Name
a) monosaccharide
b) disaccharide
c) polysaccharide
Enzymes
a) oral cavity
(mouth)
b) stomach
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.
TUBE #
A1
A2
A3
A4
A5
B1
B2
B3
B4
B5
C1
C2
C3
C4
C5
D1
D2
D3
Spectrophotometer
Reading
(Absorbance)
Factor That Was Varied
For This Group
Comments on Results
D4
D5
Name_________________________________
Pre-Lab Quiz: Enzyme Action
1. In the reaction: H-R-OH + H2O2 → 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 l00oC?
POST-LAB CRITICAL THINKING ACTIVITY - AMYLASE LAB
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 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.
SEE TOPS WEB SITE
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.
POST-LAB CRITICAL THINKING ACTIVITY
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:
Time
Iodine Test for Starch
20 seconds
Blue
40 seconds
Blue
60 seconds
Blue
80 seconds
Blue
100 seconds
Grey-blue
120 seconds
Grey-purple
Critical thinking
Draw a conclusion about the effect of dilution of the enzyme found in saliva on the
digestion of starch, and explain the results.
Amylase Lab Post Lab Activity
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
3. Restate the purpose of the lab; then design a flowchart which represents the difference
between parts A, B, C, and D.
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?
Add DI H2O,
amylopectin, and
Wait 10
Add DNS
Mix and
Hot
Analyze with
→ minutes → and titrate → Vortex → Water → Spectrophotometer
amylase to test tubes
C1-C5
solution
bath
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:
enzyme
substrate → product
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?
EQUIPMENT SKILLS CHECKLIST. Have your teacher initial next to each piece of
equipment as you demonstrate its proper use.
____________vortex
____________micropipettor
____________Brinkman pump
____________cuvette
____________loading UV-Vis spectrophotometer & scanning sample
LACTASE ENZYME LAB
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.
SAFETY: TesTape IS FOR EXTERNAL USE ONLY. IT DOES NOT BELONG
NEAR YOUR MOUTH.
WASH HANDS WITH SOAP AFTER YOU ARE DONE.
TesTape is used by people who need to test the amount of sugar in their urine because
they have diabetes. If they find sugar, it means they have too much in their blood and
they must do something about it, such as take insulin. Thus, TesTape is a very sensitive
test for glucose. Lives depend on it working well.
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.
LACTASE LAB EXTENSION
THE EFFECT OF pH ON LACTASE ACTION
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?
AMYLASE ENZYME LAB VOCABULARY BUILDERS
BG
VF
TA
NE
AQ
TZ
CW
AA
EC
RI
ND
TK
LP
RP
XQ
ABSORPTION
ACID
AMYLASE
AMYLOPECTIN
BASE
CATALYST
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DEIONIZED
DIGESTION
DNS
ENZYME
MALTOSE
PHOTO
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PRODUCT
REACTANT
SUGAR
TARTRATE
TEMPERATURE
ULTRAVIOLET