AP Biology Name________________________ Enzyme Catalysis Lab

Introduction In this lab, hydrogen peroxide will be converted into water and oxygen by an enzyme called catalase. Write the equation for this decomposition reaction in the space below: Enzymes are proteins produced by living cells. They act as catalysts in biochemical reactions. A catalyst affects the rate of a chemical reaction but is not used in the reaction. Enzymes allow cells to carry out complex chemical reactions rapidly and at relatively low temperatures. In enzyme-catalyzed reactions, the substance to be acted upon is called the substrate. The substrate binds reversibly to the enzymes active site. The temporary union creates a reduction in the activation energy needed to make the reaction occur. Enzyme function is dependent upon enzyme structure. Any environmental variable such as temperature, pH, salinity, or concentration of substrate or enzyme can affect enzyme structure and thus, function. Objectives

Observe the decomposition reaction of hydrogen peroxide by catalase Demonstrate the effects of temperature on catalase activity Learn how to establish a baseline for the amount of hydrogen peroxide in a

1.5% solution Investigate the spontaneous decomposition of hydrogen peroxide to oxygen

and water (uncatalyzed reaction or control) Use titration techniques to determine the rate of hydrogen peroxide

decomposition by enzyme catalysis Correlation to AP Biology CF Big Idea 1: EU 1D Big Idea 2: EU 2A; 2B Big Idea 4: EU 4A; 4B Background Information - Experimental Design Titration techniques will be used to measure the amount of substrate decomposed and to calculate the decomposition rate of hydrogen peroxide by an enzyme-catalyzed reaction. In this experiment the disappearance of substrate (H2O2) is measured by adding an enzyme extract that catalyzes the conversion of hydrogen peroxide to water and oxygen gas. Before all of the hydrogen peroxide is converted to product, the reaction is stopped by adding sulfuric acid. The addition of acid lowers the pH and thus denatures the enzyme stopping the enzymes catalytic activity. After the reaction is stopped, the amount of substrate remaining in the solution is measured. To measure this quantity, potassium permanganate is used.

Potassium permanganate in the presence of hydrogen peroxide and sulfuric acid reacts as follows: 5 H2O2 + 2 KMnO4 + 3 H2SO4 K2SO4 + 2 MnSO4 + 8 H2O +5 O2 Once all of the hydrogen peroxide has reacted, any more potassium permanganate added will be in excess and will not be decomposed. The addition of excess potassium permanganate causes the solution to have the permanent pink or brown color. Therefore the amount of hydrogen peroxide remaining is determined by adding potassium permanganate until the whole solution stays a pink or brown color permanently. The amount of potassium permanganate added is a proportional measure of the amount of hydrogen peroxide remaining. Check for Understanding - Write the name and molecular formula of each of the following: Enzyme:_______________________________________ Substrate:_____________________________________ Titrant:______________________________ Product(s):____________________________________ Inhibitor:____________________________ Before beginning your experiment write the question this experiment is designed to answer in the space below: Now make a prediction about the results you will obtain. Be sure to write your hypothesis as an if, then statement. Materials

1.5% Hydrogen Peroxide, H2O2 Catalase solution 1M Sulfuric Acid, H2SO4 2% Potassium Permanganate, KMnO4 Water, H2O Plastic pipettes, 1 mL Syringes 10mL Titration syringe 10mL Plastic cups (to hold each reaction) Stopwatch

Procedures A. Establishing a baseline Determining the amount of Hydrogen Peroxide in a 1.5% solution

1. Obtain three 10 mL syringes. One should be labeled S for Sulfuric Acid, one H for Hydrogen Peroxide, and one T for Transfer.

2. Dispense 10 mL of hydrogen peroxide using the H syringe into a plastic cup labeled baseline.

3. Add 1 mL of WATER to the plastic cup using a plastic pipette. 4. Swirl the cup to thoroughly mix contents. 5. Using the syringe labeled S, carefully add 10 mL of sulfuric acid to the cup.

Mix the contents by gently swirling. 6. Using the syringe labeled T, transfer 5 mL to a new plastic cup labeled

Titration. 7. Fill a titration syringe to the 10 mL marking (measure from the bottom of the

meniscus) with potassium permanganate. Note the initial reading in your data table (Table 2).

8. Slowly add one drop of potassium permanganate and swirl the solution to mix. Continue to add potassium permanganate, one drop at a time and swirling after each addition, until the solution permanently turns a light pink or brown color. The amount of potassium permanganate is proportional to the amount of hydrogen peroxide that was present in the solution.

9. Record the final volume of the syringe in your data table (Table 2). This is your baseline. Also record this value in data table 4 under baseline volume.

B. Rate of Hydrogen Peroxide Spontaneous Decomposition

1. Dispense 10mL of 1.5% hydrogen peroxide using the H syringe into a clean plastic cup labeled Control. Let the cup sit, uncovered, for 24 hr. at room temperature.

2. After 24 hours, dispense 10mL of hydrogen peroxide into a new plastic cup using the H syringe.

3. Add 1 mL of water to the cup using a plastic pipette. 4. Swirl the cup to thoroughly mix contents. 5. Using the syringe labeled S, carefully add 10 mL of sulfuric acid to the cup.

Mix the contents by gently swirling. 6. Using the syringe labeled T, transfer 5 mL to a new plastic cup labeled

Titration. 7. Fill a titration syringe to the 10 mL marking (measure from the bottom of the

meniscus) with potassium permanganate. Note the initial reading in your data table (Table 3).

8. Slowly add one drop of potassium permanganate and swirl the solution to mix. Continue to add potassium permanganate, one drop at a time and swirling after each addition, until the solution permanently turns a light pink or brown color. The amount of potassium permanganate is proportional to the amount of hydrogen peroxide that was present in the solution.

9. Record the final volume in the titration syringe in your data table (Table 3).

10. Use the formula given in Data Table 3 to calculate the amount and percent of hydrogen peroxide that spontaneously decomposed and record in your data table (Table 3)

C. Rate of Hydrogen Peroxide Decomposition by Enzyme Catalysis

1. Dispense 10 mL of hydrogen peroxide in a plastic cup labeled 10 sec. using a syringe labeled H.

2. Using a plastic pipette, add 1 mL of CATALASE solution and swirl gently for 10 Sec. to mix.

3. Using a syringe labeled S, add 10 mL of sulfuric acid to stop the reaction. 4. Using a syringe labeled T transfer 5 mL of the mixture into a new plastic

cup labeled Titrate. 5. Fill the titration syringe to the 10mL mark with potassium permanganate.

Note the initial volume in your data table (Table 4). 6. Slowly add one drop of potassium permanganate and swirl the solution to

mix. Continue to add potassium permanganate, one drop at a time and swirl after each addition, until the solution permanently turns light pink or brown. The amount of potassium permanganate added is proportional to the amount of hydrogen peroxide that was present in the solution.

7. Record the final volume in the titration syringe in your data table (Table 4). 8. Repeat this procedure for 30, 60, 90, 120, and 180 seconds using a plastic cup

labeled accordingly. 9. Use the formula given in Data Table 4 to calculate the amount of potassium

permanganate used during the titration and then the amount of hydrogen peroxide used by the enzyme catalase.

10. Record these values in your data table (Table 4). 11. Use the graph paper provide to plot the amount of Hydrogen Peroxide used

in mL vs. Time in seconds. Be sure to properly label your graph. Include both lab group and class data. Also graph SEM for the class data.

Data Enzyme activity Demonstration. Record your observations in Data Table 1. Data Table 1 Enzyme Activity Activity Observations Enzyme Activity

Effect of Extreme Temperature

Presence of Catalase in Living Tissue

Data Table 2 Establishing a Baseline for Hydrogen Peroxide Initial Syringe Reading (mL)

Final Syringe Reading (mL)

Baseline (Initial Final) (mL)

Data Table 3 Spontaneous Decomposition of Hydrogen Peroxide Initial Syringe Reading (mL)

Final Syringe Reading (mL)

Volume used after 24 hr. (mL)

Amount of H2O2 Spontaneously Decomposed (mL) (baseline-volume used after 24 hr.)

% H2O2 Spontaneously Decomposed (Amount H2O2 spontaneously decomposed/baseline X 100)

Data Table 4 Hydrogen Peroxide Decomposition by Catalase Lab Group KMnO4 used (mL) Time (seconds) 10 30 60 90 120 180 Baseline (from data table 2)

Initial Syringe Reading

Final Syringe Reading

Amount KMnO4 used (Initial Final)

Amount H2O2 used (Baseline Amount KMnO4 used)

Data Table 5 - Hydrogen Peroxide Decomposition by Catalase Class Data H2O2 Used (mL) at each time point

Lab Group Statistics 1 2 3 4 5 6 Mean H2O2

Used Standard Deviation

SEM

10 Seconds

30 Seconds

60 Seconds

90 Seconds

120 Seconds

180 Seconds

Analysis:

1. Determine the rate of this enzyme catalyzed reaction for each time interval and record your results in the table below:

Time Interval (seconds) 0 to 10 10 to 30 30 to 60 60 to 90 90 to 120 120 to 180 Rate of Reaction y x

2. When is the rate the highest? Why?

3. When is the rate the lowest? Why?

4. Explain why sulfuric acid is used as the inhibitor to the enzyme catalase.

5. Predict the effect of lowering the temperature on the rate of this reaction.

6. Using the experimental organizer provided, design an experiment to investigate the effect of temperature, pH, enzyme concentration or substrate concentration on enzyme activity.