Lab Sheet
Name: ___________________________________________________________ Date: _______________________
Amylase Race
Objective: The purpose of this investigation is to determine how reaction rate is affected by
enzyme concentration.
Background:
Enzymes are catalytic proteins that speed up the rate of chemical reactions that would otherwise
happen more slowly. A catalyst is a chemical agent that changes the rate of a reaction without
being consumed by the reaction. You have hundreds of different enzymes in each of your cells.
Each of these enzymes is responsible for one particular chemical reaction that occurs in the cell.
In this lab, you will study amylase, an enzyme that is found in the saliva of many living
organisms that use starch as a food source. Amylase breaks down the polysaccharide starch into
the disaccharide maltose. Maltose is later broken down into glucose in the intestine with the help
of digestive enzymes in the pancreas.
Normally amber yellow in color, I2KI, when exposed to starch will turn a dark purplish, black.
Because one can test for the presence of starch using I2KI, it is possible to test the effect that
concentration variation will have on the rate of amylase reaction. In order to do this, you will
make serial dilutions of amylase. These dilutions will create a range of amylase concentration
that will be tested for effectiveness in breaking down starch.
Materials:
P 1000 Micropipettor and blue tips
P 20 Micropipettor and yellow tips
5 ml pipette and pipette aide
10 test tubes and test tube rack
Multi-well test plate
1% Starch solution (stock)
1% Amylase solution (stock)
Distilled water
Wax pencil or sharpie
pH 7 buffer solution
I2KI solution
Stopwatch
Procedure:
Part A-The effect of enzyme concentration on the rate of the reaction
Preparation of Amylase dilutions:
1. Make a series of dilutions of the amylase solution in a set of 5 test tubes using Table 1 as a
guide. Use a standard 5 ml pipette. Make sure to label your test tubes, 1-5.
Lab Sheet
Table 1: Amylase Dilutions
Final quantity needed
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Concentration of final
solution
.50%
.25%
.125%
.0625%
.03125%
Milliliters (ml) of
amylase
5 ml Stock solution
5 ml Tube 1
5 ml Tube 2
5 ml Tube 3
5 ml Tube 4
Milliliters (ml) of
water
5 ml
5 ml
5 ml
5 ml
5 ml
2. Mix each test tube by gently rolling the test tube in your hands.
3. Label a new set of test tubes 1-5.
4. Transfer 2 ml from Tube 1 from step 1 into the new test tube labeled 1 using a p1000
micropipettor. Repeat his procedure for tubes 2-5.
5. At this point, you can discard the first set of test tubes.
6. Add 800 l of pH 7 buffer to each test tube from step 4. Roll the test tubes in your hands to
mix.
Preparation of test plate:
7. Add 40 l of I2KI to each well in test plate using the p20 micropipettor. Each row of the test
plate should be used for each dilution of amylase. If more wells are needed, you must refill
the test plate with I2KI after each trial. An example of a test plate is seen below.
Testing procedure:
8. Add 1 ml of 1% starch solution to tube 5. Record the time, immediately.
1. Note: Adding starch to the test tubes will begin the catalytic reaction of the breakdown of
starch to maltose by way of amylase. Therefore, the procedure becomes time sensitive at
this point.
9. Pipette out 20 l of the solution in tube 5 and add it to the first well on the test plate. (this is
time 0)10.
Lab Sheet
10. Every 20 seconds, remove an aliquot of 20 l of the solution in tube 5 and add it to the next
well in the test plate until there is no longer a reaction creating a purplish color. Record the
time. Once you have reached a well where there is no reaction, the amylase has broken down
all of the starch in the test tube, and the reaction has come to an end.
11. Repeat steps 8-10 for tubes 4-11
12. Record your data in Table 2 on the Student Worksheet.
13. Collect the data from the rest of the class and average out the seconds required for starch
digestion. Fill in the Table 3 on the Student Worksheet.
Extensions:
If time allows, students will investigate a variety of different conditions and their effect on the
enzymatic rate. Each group will be responsible for running a series of control reactions and as
well as testing one variable. We will be testing the following: substrate concentration,
temperature (of the substrate), freezing enzyme, boiling enzyme, and pH of substrate.
Student Worksheet
Name: _______________________________________________ Date: _____________
Amylase Race
IMPORTANT: Read the background and procedure then do the pre-lab questions before
performing the experiment.
Prelab Questions:
1. What color, if any, will the I2KI turn once exposed to starch?
2. What color will the I2KI turn as the amylase concentrations get more and more dilute?
3. What effect do you think amylase concentration will have on the reaction with I2KI?
Observations and Results:
Table 2: Time needed for starch digestion of amylase
Tube
1
2
3
4
5
Amylase
Concentration
(%)
.50%
.25%
.125%
.0625%
.03125%
Time required for
starch digestion
(seconds)
Student Worksheet
Collect the data from the rest of the class and average out the seconds required for starch
digestion. Fill in the following table.
Table 3: Time needed for starch digestion of amylase
Tube
1
2
3
4
5
Amylase
Time
Time
Time
Time
Time
Time
Time
Concentration required required required required required required required
(%)
for starch for starch for starch for starch for starch for starch for starch
digestion digestion digestion digestion digestion digestion digestion
(seconds): (seconds): (seconds): (seconds): (seconds): (seconds): (seconds):
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Average
.50%
.25%
.125%
.0625%
.03125%
Post lab analysis:
1. Construct a line graph showing reaction rate versus the enzyme concentration. Be sure to
label the x and y axis and title your graph.
Student Worksheet
2. What is the independent variable? What is the dependent variable?
3. What is the relationship that is observed on your graph? Explain the relationship.
4. Explain how the reaction time was measured in this experiment.
5. Is this procedure a good way to test enzyme activity? Explain your answer.
6. What do you predict would happen to the results if the procedure instructed you to make
serial dilutions of the starch (substrate) and not the amylase?
Write a paragraph describing what you learned in this lab. This paragraph should include the
purpose of the lab, a summary of the results and significance of the results. Some of the
questions above will get you started.