Total Dissolved Solids s11

Computer 2 Enzyme Action: Testing Catalase Activity (Method 1–O2 Gas Sensor)

BACKGROUND Enzymes are proteins produced by living cells, responsible for most of the chemical activities of living organisms. They act as catalysts, substances that speed up chemical reactions without being destroyed or altered during the process. Each enzyme is specific for a particular reaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure. The active site is the part of the enzyme that interacts with the substrate; any condition that blocks or changes the shape of the active site affects the activity of the enzyme. Examples of factors that affect enzyme activity are temperature, pH, and salt concentration. Most organisms have a preferred temperature range in which they survive, and their enzymes most likely function best within that temperature range. If the environment of the enzyme is too acidic or too basic, or contains a high or low salt concentration, the three dimensional structure of the enzyme may be altered. If this alteration is irreversible, the enzyme is denatured and no longer has the shape necessary for proper functioning. The enzyme used in this lab is catalase, found in animals and protists, One function of catalase within cells is to prevent the accumulation of toxic levels of hydrogen peroxide (formed as a by- product of metabolic processes).. H2O2 can be converted to oxygen and water, as follows: 2 H2O2  2 H2O + O2 Although this reaction occurs spontaneously, enzymes greatly increase the rate. A great deal can be learned about enzymes by studying the rates of enzyme-catalyzed reactions. The rate of a chemical reaction may be studied in a number of ways including:

 measuring the rate of appearance of a product (in this case, O2, which is given off as a gas)  measuring the rate of disappearance of substrate (in this case, H2O2)  measuring the gas pressure of the product as it appears (in this case, O2).

Catalase structure: Over 50 years ago physicists discovered how shining an x-ray beam on a crystal would produce a “diffraction pattern” of scattered x-rays that could be analyzed to indicate the arrangement of the atoms or molecules of the crystal. Over time, great progress was made in using “x-ray diffraction” to find out the structure of crystallized proteins. A protein is composed of a long chain of amino acid molecules that fold to produce a large molecule, and protein molecules are the basis of life. Catalase is one such molecule. X-ray diffraction studies have been used to “image” the catalase molecule, shown in the figure below: It has four subunits shown in different colors, and the spirals and ribbons are the pattern of the folded chain of proteins. There are over 500 amino acid molecules in each subunit. Near the center of each is a “heme” molecule made of an iron atom surrounded by a small structure of carbon and hydrogen atoms. It is shown to the left. The enzyme works by binding the peroxide to the heme molecule, which releases the oxygen with a chemical reaction.

Advanced Biology with Vernier 2 - 1 (O2) Computer 2

THE INVESTIGATION

In this experiment, you will use an O2 Gas Sensor to determine the rate of enzyme activity by measuring the concentration of oxygen gas formed as H2O2 is destroyed. . If a plot is made, it may appear similar to the graph shown. At the start of the reaction, there is no product, and the concentration is the same as that in the atmosphere. After a short time, oxygen accumulates at a constant rate. The slope of the curve at this initial time is constant and is called the initial rate. As the peroxide is decomposed, less of it is available to react and oxygen is produced at slower rates. The slope of the curve gradually decreases. When no more peroxide is left, O2 is no longer produced.

OBJECTIVES In this experiment, you will  Use a computer and an Oxygen Gas Sensor to measure the production of oxygen gas as hydrogen peroxide is decomposed by the enzyme catalase or peroxidase.  Measure and compare the initial rates of reaction for this enzyme when different concentrations of enzyme react with H2O2.

2 - 2 (O2) Advanced Biology with Vernier Enzyme Action: Testing Catalase Activity (O2)

 Measure the production of oxygen gas as hydrogen peroxide is decomposed by catalase or peroxidase under various conditions.

MATERIALS Vernier computer interface pH buffers computer Catalase enzyme suspension Logger Pro four 18  150 mm test tubes Vernier O2 Gas Sensor test tube rack 10 mL graduated cylinder ice Two 400 mL beakers thermometer 250 mL Nalgene bottle three dropper pipettes Bottle of 3.0% H2O2

PROCEDURE 1. Obtain and wear goggles. 2. Connect the Oxygen Gas Sensor to the computer interface. Prepare the computer for data collection by opening the file “02 (O2) Enzyme” from the Advanced Biology with Vernier folder of Logger Pro.

Part I Testing the Effect of Enzyme Concentration 3. Place four test tubes in a rack and label them 1, 2, 3, and 4. Fill each test tube with 5 mL of 3.0% H2O2 and 5 mL of water. 4. Prepare for data collection using the “data collection” screen of the experiment menu. Use the default choices or modify the time or the sampling rate. Check the default graph options on the options menu. The error bars will not be appropriate. You may want to add “point protectors” and set them up by double clicking the O2 data column to get a menu. 5. Make a trial run without enzyme. a. Pour the contents of the first test tube into a clean 250 ml Nalgene bottle.

b. Place the O2 Gas Sensor into the bottle as shown in Figure 1 below. Gently push the sensor down into the bottle until it stops. The sensor is designed to seal the bottle without the need for unnecessary force. c. When 30 seconds has passed, click to begin data collection. d. Is the data collection proceeding smoothly. Is the result what you would expect? When data collection has finished, remove the O2 gas sensor from the Nalgene bottle. Rinse the bottle with water and shake it into a paper towel e. Move your data to a stored run. To do this, choose Store Latest Run from the Experiment menu. 4. Initiate the enzyme catalyzed reaction. a. Using a clean dropper pipette, add 5 drops of enzyme suspension to test tube 2. b. Begin timing with a stopwatch or clock.

c. Pour the contents of the test tube gently into the cleaned Nalgene bottle and swirl to mix the contents

d. Place the O2 Gas Sensor into the bottle. e. When 30 seconds has passed, click to begin data collection. Does the result look reasonable?

Figure 1

5. When data collection has finished, remove the O2 gas sensor from the Nalgene bottle. Rinse the bottle with water and shake it dry into a paper towel. 6. Move your data to a stored run. To do this, choose Store Latest Run from the Experiment menu. 7. Collect data for test tubes 3 and 4:

 Add 10 drops of the enzyme solution to test tube 3. Repeat Steps 4–6.  Add 20 drops of the enzyme solution to test tube 4. Repeat Steps 4–5. 8. Using the mouse, select the initial linear region of your data on the graph. Click on the Linear Fit button, . Click and a best-fit linear regression line will be shown for each run selected. In your data table, record the value of the slope, m, for each of the four solutions in Table 1. (The linear regression statistics are displayed in a floating box for each of the data sets.) Your can drag the boxes to put them where you like.

2 - 4 (O2) Advanced Biology with Vernier Enzyme Action: Testing Catalase Activity (O2)

DATA Part I Effect of Enzyme Concentration Table 1 Test tube label Initial Slope or rate (%/min)

0 Drops 10 Drops 20 Drops

Table 2 Slopes vs.Time (%/min)

Rates 0–0.5 min 0.5–1.0 min 1.0–1.5 min 1.5–2.0 min 2.0–3.0 min 5 Drops 10 Drops 20 Drops

In your notebook draw a graph that shows how the initial slope varies with catalase concentration. Does it agree with your expectations, and if not why not? The slope becomes smaller as time goes on? Is the catalase used up or the hydrogen peroxide? How can you check your hypothesis with an experiment. What do the other members of your group think?

Part II Factors Affecting Enzyme Activity

List three factors that could possibly affect enzyme activity. Write a researchable question concerning enzyme activity. Write a procedure to test this question.

THE REMAINDER OF THIS LAB, PROCEDURES AND DATA TABLES, IS TO BE WRITTEN BY STUDENTS.

QUESTIONS (DEPENDING ON VARIABLES THAT STUDENTS CHOOSE) Effect of Temperature.

How does changing the temperature affect the rate of enzyme activity? Does this follow a pattern you anticipated?

Effect of pH How does changing the pH affect the rate of enzyme activity?

Effect of peroxide concentration

Presumably, at higher concentrations of H2O2, there is a greater chance that an enzyme molecule might collide with H2O2. If so, the concentration of H2O2 might alter the rate of oxygen production. Investigate how differing concentrations of the substrate hydrogen peroxide might affect the rate of enzyme activity.

Effect of boiling catalase

Determine the effect of boiling the catalase enzyme on the rate of the reaction.

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