BIO101_Lab06_Enzymes

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Northern Virginia Community College *

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Apr 3, 2024

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BIO 101 Lab 06: Investigating Enzyme Activity v.2021 1 BIO 101 Lab 06: Investigating Enzyme Activity **Note: Gloves are needed for this lab activity Objectives:_________________________________________________________________ Explain how enzymes facilitate chemical reactions Identify the substrate and product of catechol oxidase-catalyzed reaction Identify factors that affect enzyme activity Background:________________________________________________________________ Within an organism there are innumerable reactions occurring at any given moment. Most of these reactions are carried out by enzymes . An enzyme is a protein molecule that acts as a catalyst for a chemical reaction. A catalyst is a chemical whose presence allows a reaction to occur much quicker and more easily than it would without it. The catalyst itself comes out of the reaction unaffected. In any enzymatic reaction, the substrate attaches to the active site of the enzyme. This combination is called the enzyme-substrate complex . The substrate fits into the active site with high specificity . The interaction between substrate and enzyme causes a change to occur in the substrate molecule. Thus, the product is formed and exits the active site. If the enzyme causes the breakdown of a substrate, the reaction is an example of catabolism or a catabolic reaction. Enzymatic reactions may also combine two or more substrates into one product; this is anabolism or an anabolic reaction. Today’s lab will focus on two different enzymes: catalase and catechol oxidase . Catalase is an enzyme found in most living things. This enzyme catalyzes the conversion of hydrogen peroxide (H 2 O 2 ) into water (H 2 O) and oxygen gas (O 2 ). Conversion of hydrogen peroxide into other chemicals helps to protect cells from oxidative damage.
BIO 101 Lab 06: Investigating Enzyme Activity v.2021 2 Catechol oxidase is an enzyme found in the chloroplasts of plant cells. This enzyme catalyzes the oxidation of catechol (a chemical stored in the vacuoles of plant cells) into benzoquinone. Benzoquinone is a red-brown substance that protects a damaged plant from infection and decay. When plant cells are damaged, the catechol in the plant cell vacuoles mixes with the catechol oxidase from the chloroplasts. This results in benzoquinone forming and is one of the reasons for the “browning” of cut fruits and vegetables. We will also be examining another chemical called hydroquinone, which is structurally similar to catechol, but is not the normal substrate for the catechol oxidase enzyme. A comparison of the chemical structures of catechol and hydroquinone is provided below. Enzymes require particular conditions to function effectively. These conditions include temperature, pH, and concentration of salt in the solution. When enzymes are exposed to conditions outside of their normal range, enzyme activity is decreased and the enzymes may even become denatured . A denatured enzyme has had its three dimensional structure changed in such a way as to make it non-functional. Typically, denatured enzymes will no longer function even if the enzyme is returned to conditions in its normal range
BIO 101 Lab 06: Investigating Enzyme Activity v.2021 3 Safety:_____________________________________________________________________ Follow all standard laboratory safety procedures. Be sure to wear gloves when working with acids and bases. Lab coats are not to be worn outside of the lab. Gloves should be worn when working with acids or bases Acids and bases should be kept in the fume hood Materials:___________________________________________________________________ Experiment 1. Test tubes (5) Mushroom Banana Squash Sand Experiment 2. Test tubes (4) 1% Hydroquinone 1% Catechol Potato extract (contains catechol oxidase) Experiment 3. Test tubes (4) Ice 40 o C water bath 80 o C water bath 1% Catechol Potato extract (contains catechol oxidase) Experiment 4. Test tubes (3) 40 o C water bath (or incubator) KOH solution HCl solution 1% Catechol Potato extract (contains catechol oxidase) Procedure:__________________________________________________________________ Experiment 1 Presence of Enzymes 1. Obtain 5 small test tubes and mark each at 1 inch from the bottom of the tube. Fill each one to the mark with hydrogen peroxide.
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BIO 101 Lab 06: Investigating Enzyme Activity v.2021 4 2. Place 1 “pea - sized” piece of mushroom into 1 tube. In the second tube place 1 “pea - sized” piece of squash, in the third tube place a “pea - sized” piece of banana, and in the final tube place a pinch of sand. Leave the last tube alone as this will be your negative control. The experimental setup should look like this: 3. Observe the change (if any) in the solutions after adding the possible source of enzymes. Wait 3 minutes before recording final observations in Table 1 below. Experiments 2-4: For these experiments, you will be observing the color of the solution in each tube during the experiment. For the purposes of recording and graphing the data, use the following table to “quantify” the a mount of red color that is present in each tube. Experiment 2. Catechol oxidase enzymatic activity and effect of substrate structure on enzymatic reactions 1. Label four test tubes 2.1 2.4. Using a disposable 1.0 mL transfer pipet, add 1 mL of water to each tube. 2. Add 10 drops of water to Tube 2.1, add 10 drops of catechol to Tube 2.2, add 10 drops of catechol to Tube 2.3 and add 10 drops of hydroquinone to Tube 2.4. 3. Add 5 drops of potato extract into Tubes 2.2 and 2.3.
BIO 101 Lab 06: Investigating Enzyme Activity v.2021 5 The experimental setup should look like this: 4. Record the initial color of each tube in Table 2. 5. Place all four test tubes into a tube rack on your lab table (room temperature). Check and record color changes in Table 2 every five minutes for a total of 15 min. Experiment 3. Effect of temperature on enzymatic reaction 1. Label four test tubes 3.1 3.4. Using a disposable 1.0 mL transfer pipet, add 1 mL of water to each tube 2. Add 5 drops of potato extract into each tube. The experimental setup should look like this: 3. Place Tube 3.1 on ice, Tube 3.2 in a tube rack on your lab table (room temperature), Tube 3.3 in a 40 °C water bath (or incubator), and Tube 3.4 in a 80 °C hot water bath. Record the actual temperatures of the water baths and the room in Table 4.
BIO 101 Lab 06: Investigating Enzyme Activity v.2021 6 4. Incubate the tubes for five minutes. 5. Remove the tubes from their incubation conditions and add 10 drops of catechol to each tube. 6. Record the initial color (time zero) in Table 3. 7. Return the tubes to their designated incubation conditions (see step 2). Check and record the color of solutions in Table 3 every five minutes for 15 minutes. Experiment 4. Effect of pH on enzymatic reactions 1. Label five test tubes 4.1 - 4.5. Using a disposable 1.0 mL transfer pipet, add 1 mL of water to each tube. 2. Add 5 drops of potato extract into each tube. 3. Add 3 drops of HCl solution and 7 drops of water to Tube 4.1, 4. Add 1 drop of HCl solution and 9 drops of water to Tube 4.2, 5. Add 10 drops of water to Tube 4.3, 6. Add 1 drop of KOH solution and 9 drops of water to Tube 4.4, 7. Add 3 drops of KOH solution and 7 drops of water to Tube 4.5. 8. Add 10 drops of catechol into each tube The experimental setup should look like this: 9. Record initial color (time zero) in Table 4.
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BIO 101 Lab 06: Investigating Enzyme Activity v.2021 7 10. Place all five tubes in a tube rack on your lab bench. 11. Check and record the color of the solutions in Table 4 every five minutes for 15 minutes.
BIO 101 Lab 06: Investigating Enzyme Activity v.2021 8 BIO 101 Lab 06: Investigating Enzyme Activity Worksheet Name: __________________________ Section: ______________________ Data Analysis and Synthesis Questions: Table 1. Presence of catalase in food samples Tube Tube 1.1 Mushroom Tube 1.2 Squash Tube 1.3 Banana Tube 1.4 Sand Tube 1.5 Control Observations Did it contain enzymes that react with hydrogen peroxide? (yes/no) Table 2. Color (amount of “redness” from 0 -5) of solution observed in each tube as a result of catechol oxidase enzymatic activity using different substrates Time (in minutes) Tube 2.1 H 2 O Tube 2.2 Catechol Tube 2.3 Catechol + Enzyme Tube 2.4 Hydroquinone + Enzyme 0 (initial color) 5 10 15
BIO 101 Lab 06: Investigating Enzyme Activity v.2021 9 Graph 1. Bar Graph of Enzyme Activity (amount of “redness” at 15 minutes) versus the contents of each tube Table 3. Color (amount of “redness” from 0 -5) of solution observed in each tube as a result of enzymatic reactions at different temperatures Time (minutes) Tube 3.1 Tube 3.2 Tube 3.3 Tube 3.4 _____°C _____°C _____°C _____°C 0 5 10 15
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BIO 101 Lab 06: Investigating Enzyme Activity v.2021 10 Graph 2. Line Graph of Enzyme Activity (amount of “redness” at 15 minutes) versus Temperature
BIO 101 Lab 06: Investigating Enzyme Activity v.2021 11 Table 4. Color (amount of “redness” from 0 -5) of solution observed in each tube as a result of enzymatic activity in different pH conditions Time (minutes) Tube 4.1 pH 4 Tube 4.2 pH 6 Tube 4.3 pH 7 (Control) Tube 4.4 pH 8 Tube 4.5 pH 10 0 5 10 15 Graph 3. Bar Chart of Enzyme Activity (amount of “redness” at 15 minutes) versus pH
BIO 101 Lab 06: Investigating Enzyme Activity v.2021 12 1. In Experiments 2, 3, and 4, what is the source of enzyme? 2. In Experiment 2, when comparing Tube 2.3 and Tube 2.4: a. Which tube changed color from light yellow to red-brown the fastest? b. Explain the results. 3. Based on the results of Experiment 3: a. What is the optimum temperature for the function of catechol oxidase? b. Explain what happened to the enzyme in the 80⁰C water bath .
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BIO 101 Lab 06: Investigating Enzyme Activity v.2021 13 4. Based on the results of Experiment 4: a. Does catechol oxidase work efficiently at low pH? b. Explain why squeezing lemon juice on a cut potato can slow down the tissue browning of the potato. Instructor signature_______________________________