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 10 cm from bottom of each test tube and mark.
3. Fill to the 10 cm mark with deionized water.
4. Place test tubes in test tube rack.
Tube 1:
1. Add 10 drops of enzyme and 10 drops of substrate.
2. Using Parafilm, shake the test tube several times.
3. Record color in Table 1 at the end of 5 minutes. (This is
the end color to be expected for this reaction.)
Tube 2:
1. Add 10 drops of substrate only.
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the end color.
Tube 3:
1. Add 10 drops of enzyme and 10 drops of sucrose.
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the end color.
Table 1: Enzyme Specificity
Test
Contents
Tube
1
2
3
Ending Color
1
Record colors
as the spectral
transmission at
470 nm.
Questions:
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.
B. Effect of Temperature on Enzyme Activity
1. Number 3 test tubes and mark to identify ownership by lab group.
2. Measure 10 cm from bottom of each test tube and mark.
3. Fill to the 10 cm mark with deionized water.
4. Place each test tube in its designated environment:
Test tube 1
Place in ice bath (5°C)
Test tube 2
Place at room temperature (22°C)
Test tube 3
Place in hot water bath (80°C)
5. After 15 minutes, while still in their respective environments, add 10
drops of substrate and 10 drops of enzyme to each tube.
6. Allow each tube to remain in its environment for 5 minutes.
7. After 5 minutes, note the end color of each tube and record in
Table 2.
Table 2: Effect of Temperature and Enzyme Activity
Test
Environment
Ending Color
Tube
1
Ice bath
2
Room
3
Hot water bath
Questions:
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. Number 3 test tubes.
2. Measure 10 cm from bottom of each test tube and mark.
3. Fill to the 10 cm mark with deionized water.
4. Place test tubes in test tube rack.
Tube 1:
1. Add 3 drops of enzyme and 10 drops of substrate.
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the color.
Tube 2:
1. Add 9 drops of enzyme and 10 drops of substrate.
2
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the end color.
Tube 3:
1. Add 27 drops of enzyme and 10 drops of substrate.
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the end color.
Table 3: Effect of Enzyme Concentration on Enzyme Activity
Test
Amount of Enzyme
Ending Color
Tube
1
2
3
Questions:
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. Number 3 test tubes.
2. Measure 10 cm from bottom of each test tube and mark.
3. Fill to the 10 cm mark with deionized water.
4. Place test tubes in test tube rack.
Tube 1:
1. Add 3 drops of substrate and 10 drops of enzyme.
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the end color.
Tube 2:
1. Add 9 drops of substrate and 10 drops of enzyme.
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the end color.
Tube 3:
1. Add 27 drops of substrate and 10 drops of enzyme.
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the end color.
Table 4: Effect of Substrate Concentration on Enzyme Activity
Test
Amount of Substrate
Ending Color
Tube
1
2
3
3
Questions:
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?
E. Effect of Inhibitors on Enzyme Activity
1. Number 3 test tubes.
2. Measure 10 cm from bottom of each test tube and mark.
3. Fill to the 10 cm mark with deionized water.
4. Place test tubes in test tube rack.
Tube 1:
1. Add 10 drops of enzyme and 10 drops of substrate.
2. Using Parafilm, shake the test tube several times.
3. Observe for 5 minutes and record the end color
Tube 2:
1. Add 10 drops of enzyme and 10 drops of substrate.
2. Add 1 drop of inhibitor.
3. Using Parafilm, shake the test tube several times.
4. Observe for 5 minutes and record the end color.
Tube 3:
1. Add 10 drops of enzyme and 10 drops of substrate.
2. Add 20 drops of inhibitor.
3. Using Parafilm, shake the test tube several times.
4. Observe for 5 minutes and record the end color.
Table 5: Effect of Inhibitors on Enzyme Activity
Test
Amount of Inhibitor
Ending Color
Tube
1
2
3
Questions:
1. What effect does the increase of the inhibitor have on enzyme action?
Using Excel, make bar graphs of the data from the preceding tables.
Turn in a standard report, including your graphs, answers to
questions, and conclusions about the general behavior of enzymes
with the observed variables.
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