AP LAB TWO: ENZYME CATALYSIS

OVERVIEW

In this lab you will:

1.  observe the conversion of hydrogen peroxide (H2O2) to water and oxygen gas by the enzyme catalyze, and

2.  measure the amount of oxygen generated and calculate the rate of the enzyme-catalyzed reaction

Before doing this lab you should understand:

·  the general functions and activities of enzymes;

·  the relationship between the structure and function of enzymes;

·  the concept of initial reaction rates of enzymes;

·  how the concept of free energy relates to enzyme activity;

·  that changes in temperature, pH, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions; and

·  catalyst, catalysis and catalase

OBJECTIVES

After doing this lab you should be able to:

·  measure the effects of changes in temperature, pH, enzyme concentration, and substrate concentration on reaction rates of an enzyme-catalyzed reaction in a controlled experiment; and

·  explain how environmental factors affect the rate of enzyme-catalyzed reactions.

EXERCISE 2A: Catalase Activity Demonstration

The teacher will demonstrate the catalytic activity of the potato catalase. This enzyme will break hydrogen peroxide into water and oxygen gas. Some of the potato catalase will be kept at room temperature while some will be heated in a water bath. Both hydrogen peroxide and water will be added to samples of these two types of enzymes.

In your notebook, make a prediction of what will occur in the different types of enzymes when water and hydrogen peroxide is added to samples of each. At the end of the demonstration, state whether your prediction was correct or incorrect and make an explanation of what happened based on your understanding of enzymes, substrates and chemical reactions.

EXERCISE 2B: Differing Concentrations of Enzyme and its effect on its activity

To determine the affect of Catalase concentration on enzyme activity

Procedure

  1. Obtain “100%” catalase extract from teacher.
  2. Prepare enzyme dilutions as shown in following table 2.1. Put into labeled beakers. Remember to rinse graduated cylinders before and after use to avoid contamination.
  3. Obtain the 3% hydrogen peroxide solution.
  4. Pour 60 ml of the hydrogen peroxide solution into a clean beaker and label as “reaction beaker.”
  5. Pick up a filter paper disk with clean forceps. Using the for forceps, dunk the disk in your enzyme extract for FIVE (5) seconds, until the disk is uniformly moistened but not beaded with shiny drops of liquid.
  6. Drain filter paper disk on paper towel for 3 seconds to remove excess enzyme from the disk
  7. Using forceps, place the enzyme moistened disk onto the bottom of the “reaction beaker” containing the hydrogen peroxide.
  8. Record the number of seconds that it takes for the disk to reach the top of the liquid in a flat orientation. Be precise in your timing and recording. Take observations of the disk and its travel upward.
  9. Remove the disk and discard it.
  10. Obtain another disk and repeat steps 5-9 exactly as done above for the other 4 trials.
  11. Repeat steps 1-10 for other concentrations of enzymes.
  12. Clean up all materials.

Table 2.1

Enzyme Extract Concentration / Volume of Enzyme / Volume of Water
20% / 8 ml / 32 ml
40% / 16 ml / 24 ml
60% / 24 ml / 16 ml
80% / 32 ml / 8 ml
100% / 40 ml / 0 ml

Table 2.2: Change in Enzyme Concentration Results – Individual data

Enzyme Concentration / Trial #1
(seconds) / Trial #2
(seconds) / Trial #3
(seconds) / Trial #4
(seconds) / Trial #5
(seconds) / Team Average

Table 2.3: Change in Enzyme Concentration Results – Class Data

Average Time (sec) for Enzyme Filter Paper to Rise
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 / Total / Class Average / Rate
(1/Time)
20% Enzyme
40% Enzyme
60% Enzyme
80% Enzyme
100% Enzyme

13.  Graph the results for both your individual data and the class average. Be sure to place the independent variable on the horizontal (x) axis and the dependent variable on the vertical (y) axis (Rate of reaction). Label the Graph 2.1 and 2.2 AND provide analysis and title for the graphs.

EXERCISE 2C: Effect of Substrate Concentration on Enzyme Activity

In this exercise you will determine the affect of varying substrate concentration on the rate at which an enzyme can work.

Procedure

1.  Obtain stock hydrogen peroxide and prepare the following solutions as shown in Table 2.4 and put in labeled beakers.

2.  Obtain 60% catalase solution from teacher

  1. Pick up a filter paper disk with clean forceps. Using the for forceps, dunk the disk in your 60% enzyme extract for FIVE (5) seconds, until the disk is uniformly moistened but not beaded with shiny drops of liquid.
  2. Drain filter paper disk on paper towel for 3 seconds to remove excess enzyme from the disk
  3. Using forceps, place the enzyme moistened disk onto the bottom of the “reaction beaker” containing the specific concentration of hydrogen peroxide you are working on.
  4. Record the number of seconds that it takes for the disk to reach the top of the liquid in a flat orientation. Be precise in your timing and recording. Take observations of the disk and its travel upward.
  5. Remove the disk and discard it.
  6. Repeat this procedure (steps 3-7) for all of the concentrations of hydrogen peroxide.
  7. Clean up all materials

Table 2.4

Substrate Extract Concentration / Volume of Hydrogen Peroxide / Volume of Water
2.0% / 40 ml / 20 ml
1.5% / 30 ml / 30 ml
1.0% / 20 ml / 40 ml
0.8% / 16 ml / 44 ml
0.6% / 12 ml / 48 ml
0.3% / 6 ml / 54 ml

Table 2.5: Change in Substrate Concentration Results – Individual data

Enzyme Concentration / Trial #1
(seconds) / Trial #2
(seconds) / Trial #3
(seconds) / Trial #4
(seconds) / Trial #5
(seconds) / Team Average

Table 2.6: Change in Substrate Concentration Results – Class Data

Average Time (sec) for Enzyme Filter Paper to Rise
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 / Total / Class Average / Rate
(1/Time)
2% [Substrate]
1.5% [Substrate]
1.0% [Substrate]
0.8% [Substrate]
0.6% [Substrate]
0.3% [Substrate]

10.  Graph the results for both your individual data and the class average. Be sure to place the independent variable on the horizontal (x) axis and the dependent variable on the vertical (y) axis (Rate of reaction). Label the Graph 2.1 and 2.2 AND provide analysis and title for the graphs.