The Effects of Selected Variables on Enzyme Activity
Enzymes are protein molecules that speed up chemical
reactions. Enzymes possess a 3-dimensional shape that will
bond only to a specific reaction molecule or molecules. The
molecule to which an enzyme bonds is called the substrate
molecule. Substrate molecules bond to a region of the
enzyme called the active site. The active site is where the
chemical reaction occurs between the enzyme and substrate
molecules.
Objective…
Demonstrate an enzymatic reaction and determine the effects of selected variables on the rate of
reactions involving enzymes.
Materials and Method…
A. Enzyme Specificity
1. Number 3 test tubes.
2. Measure 8 cm from bottom of each test tube and mark with wax pencil.
3. Fill to the 8 cm mark with deionized water (the gray faucet up front).
4. Place test tubes in test tube rack.
5. Perform the following procedures for the three tubes:
Record colors as the spectral
transmission at 470 nm.
You’ll have to pour a sample
from each tube into the
smaller spec tubes. The %
transmission is actually how
much light is able to pass
through the liquid—so the
lower the number the less
light, or the more intense the
color. The more intense the
color, the greater the
enzyme activity.
Tube 1: Add 10 drops of enzyme and 10 drops of substrate.
a) Using parafilm to seal, shake the test tube several times.
b) After exactly 10 minutes (work quickly), pour some of the liquid into the smaller
tube (about ¾ full) and place in spectroscope.
c) Record % transmission.
Tube 2: Add 10 drops of substrate only. Repeat steps a,b,c.
Tube 3: Add 10 drops of enzyme and 10 drops of sucrose. Repeat steps a,b,c.
Table 1: Enzyme Specificity
Test
Tube
1
2
3
Contents
% Transmission
Questions: (include these in your report)
1. Which of the three test tubes is the control group?
2. Which of the three test tubes demonstrated enzyme specificity?
3. Explain how this experiment shows that enzymes are specific to a substrate.
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B. Effect of Temperature on Enzyme Activity
1. Rinse the tubes from the following activity and fill to the mark with deionized water.
2. Place each test tube in its designated environment:
Tube 1: Place in ice bath (5°C)
Tube 2: Place at room temperature (22°C)
Tube 3: Place in hot water bath (80°C)
3. After 5 minutes, while still in their respective environments, add 10 drops of substrate and
10 drops of enzyme to each tube. Swirl to mix.
a) After exactly 10 minutes (work quickly), pour some of the liquid into the smaller
tube (about ¾ full) and place in spectroscope.
b) Record % transmission.
Table 2: Effect of Temperature and Enzyme Activity
Test
Environment
Tube
1
Ice bath
2
Room
3
Hot water bath
% Transmission
Questions: (include these in your report)
1. Which test tube demonstrated the fastest reaction rate?
2. Why did the test tube you selected demonstrate the fastest reaction rate.
C. Effect of Enzyme Concentration on Enzyme Activity
1. Rinse tubes and fill to the 8 cm mark with deionized water.
2. Perform the following procedures for the three tubes:
.
Tube 1: Add 3 drops of enzyme and 10 drops of substrate.
a) Seal and shake the tube several times.
b) After 10 minutes, record the % transmission.
Tube 2: Add 9 drops of enzyme and 10 drops of substrate. (repeat steps a and b)
Tube 3: Add 27 drops of enzyme and 10 drops of substrate. (repeat steps a and b)
Table 3: Effect of Enzyme Concentration on Enzyme Activity
Test
Amount of Enzyme
Tube
1
2
3
2
% Transmission
Questions: (include these in your report)
1. Which test tube exhibited the fastest reaction rate?
2. Why did the test tube you selected in the fastest reaction rate?
D. Effect of Substrate Concentration on Enzyme Activity
1. Rinse tubes and fill to the 8 cm mark with deionized water.
2. Perform the following procedures for the three tubes:
Tube 1: Add 10 drops of enzyme and 3 drops of substrate.
a) Seal and shake the tube several times.
b) After 10 minutes, record the % transmission.
Tube 2: Add 10 drops of enzyme and 9 drops of substrate. (repeat steps a and b)
Tube 3: Add 10 drops of enzyme and 27 drops of substrate. (repeat steps a and b)
Table 4: Effect of Enzyme Concentration on Enzyme Activity
Test
Amount of Substrate
Tube
1
2
3
% Transmission
Questions: (include these in your report)
1. Which test tube had the fastest reaction rate?
2. Why did the test tube you selected in Question 1 exhibit the fastest reaction rate?
Make sure each group member has a copy of the results. Using Excel,
make separate bar graphs of the data from the preceding tables. Submit a
standard report, including one of your graphs, answers to questions,
and conclusions about the general behavior of enzymes with the
observed variables.
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