Name: _____________________________________ Per: ______
ENZYME- LAB
BACKGROUND
Enzymes are molecules that belong to the _________________________
macromolecule group. They catalyze Rx which means they ____________ up the rate of a
chemical reactions but are not ________________________during the reaction. At the end of
the reaction catalysts can be recovered in their original form. Each metabolic Rx in the body
requires its own specific enzyme, because each enzyme fits only one specific substrate a
phenomenon that is commonly referred to as the ___________________ mechanism. For
example the function of amylase enzymes in our saliva and pancreatic secretions is to
hydrolyze the starch in our diets. Other examples of enzymes are __________________ which
break the esterbonds of triglycerides or the enzyme _____________________ which breaks the
bond between glucose and fructose in the ____ saccharide sucrose.
The three-dimensional tertiary structure of the protein plays a very important role in the
function of enzymes. The tertiary/quaternary structure helps maintain the shape of the protein
such that the protein is able to recognize and bind a select group of molecules called
____________________ The region of the enzyme responsible for binding the substrate and
carrying out the reaction is called the ___________________. The active site of an enzyme
has a specific three-dimensional shape that allows only one or only a small number of substrate
molecules to bind, making enzymes highly specific.
An enzyme with a substrate bound to the active site is called an
______________________________. The activity of the enzyme, or how fast the enzyme
works, is strongly influenced by reaction conditions such as the enzyme concentration,
temperature, pH and the presence of inhibitors. Most enzymes in the body function optimally
physiological conditions: pH of 7.4, temperature of 38 Celcius (98.4 0F) Under these conditions,
the enzyme has the perfect _________________________--shape to bind the substrate.
In this lab you will test the activity of the enzyme Amylase.
Comlete the reaction below by indentifying the substrate and the product of Amylase
Amylase
Testing for enzyme activity
In order to test for enzyme activity, you need to be able to measure concentrations of substrate
and product.
What is the test for starch and how does a positive and negative test look like?
What is the test for glucose and how does a positive and negative test for glucose looks like?
Enzyme Concentration: The rate of an enzyme reaction also depends on the enzyme
concentration, the _________ enzyme molecules are present the _______ the reaction goes.
Temperature: The temperature at which an enzyme is most active is called the optimum
temperature. Above and below the optimum temperature the activity of the enzyme is reduced.
Most enzymes in the human body work best at around the temperature of ________________
Some types of bacteria that are found near geothermal vents, where the temperature is
significantly higher and therefore have enzymes that function optimally in these ranges. If the
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Name: _____________________________________ Per: ______
enzymes experiences temperatures significantly higher than its optimal temperature, the
enzyme _________________ which means it starts to break down.
pH: Enzymes are most active at their optimum pH. At pH values above or below the optimum
pH the activity of the enzyme is reduced. Most cellular enzymes have an optimum pH close to
the physiological pH of 7.4. In contrast enzymes that work in the stomach have optimum values
around pH 1 at which they obtain a perfect 3D structure. Interesting enough the stomach is the
part of the digestive system to digest exclusively the proteins found in our food. The acid
__________________ the food proteins which is the first step of breaking them down
Inhibitors: Molecules that prevent the active site from binding the substrate and cause
enzymes to lose activity are called inhibitors. Inhibitors that compete with the substrate for the
active site are called ____________________________ inhibitor. Inhibitors that bind to a site
other than the active site, but still changes the active site conformation and prevents substrate
binding are called a _______-competitive inhibitors.
PROCEDURE
The goal of this laboratory is to introduce you to some of the concepts relating to the activity of
enzymes. You will observe how enzyme concentration, temperature, pH and inhibitors affect the activity
of the enzyme amylase. Amylase is present in our saliva as well as our pancreatic secretions and is
responsible for hydrolyzing the carbohydrate amylose (a component of starch) in our diet. Amylase will
hydrolyze amylose to give smaller polysaccharides, disaccharides such as maltose, and given sufficient
time the monosaccharide glucose. Eventually amylase will convert all the amylose to glucose.
You will determine the activity of amylase by testing for the presence of amylose in a sample of
starch using iodine. Iodine produces a deep blue-black color with amylose. As the amylase enzymes
breaks down the amylose in the starch the iodine test will become less and less positive. When all of the
amylose has been converted to glucose the iodine test will be negative and only the red or gold color of
the iodine reagent will be seen. Based on the color with the iodine reagent and how fast the blue-black
color is lost you can determine the activity of amylase. You will also use the Benedict’s test to detect the
appearance of the hydrolyzed product, glucose.
You will use the iodine test to investigate how enzyme concentration, temperature, pH and an
inhibitor affect the activity of amylase.
Constant temperature water baths.
Type of Bath
Temperature of Water Bath
Beaker with Boiling hot water
100°C
Adjustable Water bath
37°C
Beaker with Ice water
0°C (or lower than 5°C)
Starch solution. Place 20 mL of 1% starch solution in a 50 mL beaker to use as needed
Iodine solution. Use iodine dropper bottles.
Amylase. Store 10 mL of amylase solution provided by the instructor in a Styrofoam ice box.
Reference test. Place 5-6 drops of starch solution in an empty well of your plate and add 1-2
drops of iodine reagent. The reaction with starch should give a deep blue-black color.
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Visual Color Reference. As you proceed with each experiment, you will check enzyme activity
by adding iodine to the starch mixture. When enzyme activity is high, the time required for the
starch to hydrolyze will be very short. When the enzyme is slowed down or inactive, the blueblack color will be seen for a longer time. By observing the disappearance of starch, you can
assess the relative amount of enzyme activity as follows:
Iodine Test Color
Amount Starch Remaining
Enzyme Activity Level
All
None (0)
Gray/Brown
Most
Low (1)
Light brown
Some
Moderate (2)
Gold
None
High (3)
Dark blue black
Use clean pipettes when transferring solutions to avoid cross-contaminating samples.
PROCEDURE A: EFFECT OF ENZYME CONCENTRATION, CONSTANT TEMPERATURE
AT 37 C
Materials: Micro-centrifuge tubes, 37°C water bath, 400 mL beaker, floating rack, plastic 24 well plate,
amylase solution, pipettes, 1% starch (buffered pH 7.0), and iodine reagent.
1.
2.
3.
4.
Place 1 mL of 1% starch solution in four separate micro-centrifuge tubes labeled 1-4.
Place 1.5 mL of amylase solution in a fifth tube.
Place all five tubes in a 37°C water bath for 5 minutes.
While the solutions are heating, prepare your well plate to check enzyme activity. Add 12 drops of iodine solution to 4 empty wells in 3 adjacent rows (3x 4=12)
5. At the end of the 5 minute warming period add the respective drops of warmed amylase
(Table below!) from tube 5 to the labeled 4 starch containing tubes. Cap tube after
adding the amylase and shake to mix.
6. Then immediately transfer four drops of each reaction mixture (use a clean pipette with
each transfer) to the first rows of wells containing iodine reagent.
Tube Number
Amount 1% Starch
Amount Warmed Amylase
1
1 mL
0 drops (control)
2
1 mL
3 drops
3
1 mL
6 drops
4
1 mL
10 drops

RETURN THE MICRO-CENTRIFUGE TUBES IMMEDIATELY TO THE 37°C WATER
BATH.
 AT 5 AND 10 MINUTES REMOVE 4 DROPS FROM EACH TUBE 1-4 AND REPEAT
THE IODINE TEST.FOR ROW 2 AND 3. RECORD YOUR RESULTS.
Record your observations for each sample. Use the visual color reference to assess the
enzyme activity.
Wash your well plate!!!
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RESULT A: EFFECT OF ENZYME CONCENTRATION
Observations – color produced by iodine test and activity level
A.1. Time
No amylase
Color
Level
3 drops amylase
Color
Level
6 drops amylase
Color
Level
10 drops amylase
Color
Level
0 min
5 min
10 min
Graph A: Enzyme Concentration: Enzyme Activity at 10 minutes vs. enzyme concentration in
drops. Have 3 lines for the three incubation times all in one graph. Label your axis.
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PROCEDURE B: EFFECT OF TEMPERATURE
Materials:
Micro-centrifuge tubes, water baths: ice (0°C), warm (37°C) and boiling (100°C), 400 mL
beaker (2), floating racks, hot plate, plastic 24 well plate, pipettes, buffer solution (pH 7), amylase
solution, 1% starch, and iodine reagent.
1. Place 0.75 mL of 1% starch solution in three separate micro-centrifuge tubes.
2. Place one tube in the boiling water bath, one tube in the 37°C water bath, and one tube
in the ice bath.
3. Add 6 drops of buffer solution pH 7 and 5 drops of amylase solution to three other tubes,
and place one each in the three water baths.
4. Let them remain in the water baths for about 10 minutes to allow the solutions to reach
bath temperature. (You should have 6 tubes, 3 containing 1% starch and three
containing amylase. Each Bath should have two tubes 1 each of the starch and
amylase.)
For each temperature bath, proceed as follows:
a. Remove the micro-centrifuge tubes, and pour the starch solution into the amylase solution, cap
the tube and shake well to mix. Return the mixture to the same temperature bath and incubate
for 5 min
b. Add 1-2 drops of iodine reagent to 3 empty wells of row 1 in your well plate. After 5 minutes,
transfer four drops of the mixture (use a clean pipette each time) to a well containing iodine
reagent. Record the color and activity level.
RESULTS B: EFFECT OF TEMPERATURE ON ENZYME ACTIVITY
Temperature
Color
Level
0 0C
37 0C
100 0C
Graph B: Enzyme activity at different temperatures. Label your axis.
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PROCEDURE C. EFFECT OF PH ON ENZYME ACTIVITY
Materials:
Micro-centrifuge tubes, 37°C water bath, 400 mL beaker, floating rack, plastic 24 well
plate, pipettes, buffers (pH 2,4, 7, and 10), amylase solution, 1% starch, and iodine reagent.
1. Place 6 drops of buffer solutions of pH 2, 4, 7, and 10 in four separate, labeled microcentrifuge tubes.
2. Add 3 drops of amylase solution in each. In four other tubes, place 0.75 mL of 1%
starch solution.
3. Place all the tubes in a 37°C water bath for about 5 minutes.
4. Pour each of the 1% starch solutions into a separate buffer-amylase tube. Cap the tube
and shake well to mix. Return the mixtures to the 37°C water bath. Note the time.
5. Add 1-2 drops of iodine reagent to 4 empty wells in row 2 in your well plate. After 10
minutes, remove four drops of each reaction mixture (use a clean pipette each time) and
add to a well containing iodine reagent. Record your observations and the enzyme
activity level for each reaction mixture.
RESULTS C: EFFECT OF PH ON ENZYME ACTIVITY
pH
2
4
7
10
Color
Level
Graph C: Enzyme activity level vs. pH. Label your axis.
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PROCEDURE D.
INHIBITION OF ENZYME ACTIVITY
Materials:
Micro-centrifuge tubes, 37°C water bath, 400 mL beaker, floating rack, plastic 24 well
plate, pipettes, 1% AgNO3 and 1% NaCl solutions, 95% ethanol, amylase solution, 1% starch, and iodine
reagent.
1. In one micro-centrifuge tube, place 3 drops of amylase solution and 10 drops of pH 7
buffer solution.
2. In a second micro-centrifuge tube, place 3 drops of amylase solution and 10 drops of 1%
NaCl.
3. In a third tube, place 3 drops of amylase solution and 10 drops of ethanol.
4. In a forth tube, place 3 drops of amylase solution and 10 drops of 1% AgNO3. Label
each tube.
5. Place 1 mL of 1% starch solution in four other tubes.
6. Place all eight tubes in a 37° water bath for 10 minutes.
7. Pour the 1% starch solution into the amylase solution, cap the tube and shake well to
mix.
8. Return the tubes to the 37°C water bath for 15 minutes.
9. Add 1-2 drops of iodine reagent to 4 empty wells of row 3 in your well plate.
10. Remove four drops of each reaction mixture (use a clean pipette each time) and add to a
well containing iodine reagent. If the AgNO3 well loses its color after transferring it to a
well containing iodine reagent, add 1-2 extra drops of iodine reagent. Record your
observations and the enzyme activity for each of the reaction mixtures.
RESULTS D: INHIBITION OF ENZYME ACTIVITY
D.1. Observations – color produced by iodine test and activity level
Compound
Color
Level
Buffer pH 7
NaCl
Ethanol
AgNO3
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QUESTIONS
(1). In part A you investigated the activity of amylase while increasing the enzyme
concentration. Describe the effect of enzyme concentration on enzyme activity.
(2). In parts B and C you investigated the activity of amylase at different temperatures and at
different pH values. Based on the graphs you drew what was the optimal temperature and pH
of amylase?
(3). Pepsin is an enzyme that helps break down proteins in our stomach. The pH of our
stomach is 2. What do you think is the optimum pH of pepsin?
(4). In part D you investigated the activity of amylase in the presence of three different
compounds. Which compound do you think was an inhibitor of enzyme activity?
PRE-LABORATORY QUESTIONS
(1). Research and draw graphs (one each) to show how temperature and pH affect the activity
of an enzyme (i.e., reaction rate). Your graphs should have correctly labeled axis. On your
graph indicate where you would expect to observe the optimum temperature and optimum pH
for the enzyme.
(2). Explain how high temperatures decrease enzyme activity as does extreme pH values
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