Science Experimeny

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ENZYME LAB

Introduction- Enzymes are proteins that speed up the rate of reactions in living things. In this lab, we will perform four experiments exploring the way enzymes work.

PART A: pH SPECIFICITY

Every enzyme has a specific pH at which it works best. In this section, you will determine which pH is best for the enzyme, catalase.

Living tissues produce the enzyme catalase, which is able to break down hydrogen peroxide into oxygen gas and water. The reaction is shown below:

H2O2 O2 + H2O
Cells make hydrogen peroxide as a normal product of metabolism. But it is toxic to the cell if it builds up. Therefore, all cells produce catalase to get ride of hydrogen peroxide. In the following experiment, we will use liver as a source of catalase.

Before you do the procedures below, set up a lab paper with overall lab title, sections lab title, propose and hypothesis.

Procedures: 1. Get 3 test tubes and label them 1 through 3 with a pen. Place all tubes in a rack.. 2. Get 3 pieces of raw liver (how gross) and place 1 piece in each test tube. 3. Using a graduated cylinder, add 5 ml of distilled water to tube 1. 4. Put on your goggles. Using the graduated cylinder labeled acid, add 5ml of hydrochloric acid (HCl) to tube 2. Be careful. 5. Using the graduated cylinder label base, add 5ml of sodium hydroxide (NaOH) to tube 3. 6. Record the contents of all tubes in a data chart on your lab paper. 7. Wait 3min. 8. You will now use a graduated cylinder labeled H2O2 to add 5ml of hydrogen peroxide to each tube. Be sure to bring the hydrogen peroxide to your tube in the rack. Never hold your test tube in your hand while pouring in the hydrogen peroxide. Be prepared for overflow. 9. Check for the production of gas in the data charts for each tube, using the terms none, fair, or many. 10. Clean up procedures: -Throw all liver in the trashcans. - Empty liquids down the drain, rinse, and place tubes in dirty bin in front of the room.
Answer the questions below on your lab paper: 1. What is the substrate in this experiment? What are the products of reaction? What is the name of the enzyme? What is the source of the enzyme? 2. Why did we look for bubbling in the test tube? What does bubbling tell you? 3. Based on your data. At what pH does catalase enzyme work the best? Explain how you know. 4. What affects does too much acid or too much base have on an enzyme? Be specific.

PART B: TEMPERATURE SPECIFICITY

Most enzymes have a certain temperature range to which they work best. Outside of the temperature range, an enzyme’s structure may be destroyed, rendering it ineffective. In this experiment, we will determine the effect that boiling temperatures have on the enzyme catalase. We will compare the gas production with boiled liver versus raw, room temperature liver.

Before you start the lab, write the section title, purpose and a hypothesis on your lab paper.

Procedures: 1. Get 2 clean test tubes and label them 4 and 5. 2. To tube 4 add a piece of raw liver. 3. To tube 5 add a piece of cooked liver. 4. Add 5ml of distilled water to each tube. 5. Add 5ml of hydrogen peroxide to each tube. 6. Record the contents of each tube in your data chart, as well as the relative amounts of bubble production. 7. Clean up as instructed in section A.

Answer: 1. Based on your data, what effect does boiling have on the effectiveness of catalase enzyme? Explain how you know. 2. Suppose you had prepared another test tube with frozen raw liver, distilled water, and hydrogen peroxide. What affects do you think very low temperatures might have on the catalase enzyme? 3. At what approximate temperatures does catalase enzyme work the best? 4. What affects does temperature outside the normal optimum have on the catalase enzyme? Be specific.

PART C: SURFACE AREA AND ENZYME ACTION

In order of an enzyme to be most effective, it most be exposed to its substrate. In this experiment, you well use a potato as a source of catalase. 1 test tube will contain the enzyme within a solid potato cub. The other test tube will contain the enzyme in a masked raw potato.

Before toy start this section, write the section title, purpose and a hypothesis on your lab paper.
Produces:
1. Get 2 new test tubes and label them 6 and 7. 2. to tube 6, add a whole raw potato cube. 3. To tube 7, add an equal amount of raw, mashed potatoes. 4. Add 5ml of distilled water to each tube. 5. Add 5ml of hydrogen peroxide to each tube. 6. Record the contents of the tubes in your data chart. 7. Record the relative amounts of bubble production in each tube.

Answer:
1.Which form of the potato, raw or mashed, had more surface area to make catalase enzyme more available?
2. Which form of the potato showed more enzyme action-taking place? Why?

PART D: SUBSTRATE SPECIFICITY
Enzymes are specific to respect to the substrate they will react with. The active site of an enzyme fits its substrate like a key fits only a specific lock.. Your body has 1,000 of different enzymes, each capable of catalyzing 1 specific reaction. In his section you will compare the effect 2 different enzymes, amylase and pepsin, have on the substrate, starch.

Before you set up the lab, write the section title and purpose on your lab paper, then answer these questions. 1. Look up amylase and pepsin in the digestion chapter of your text. What substrate does amylase react with? What substrata does pepsin react with? 2. After reading the procedures below, write a hypothesis stating which test tube you think will show the digestion of starch by enzymes? Why? 3. Why do we incubate the test tubes at body temperature? 4. What is the purpose of test tube 10? Have you set up a controlled experiment? What is the variable?

Procedure:
Day 1: 1. Get 3 test tubes and label them 8, 9 and 10. Put 1 person initials on each tube. 2. Using a clean graduated cylinder, add 5 ml of starch suspension to each tube. Rinse the graduated cylinder several times with water. 3. Add 2 ml of pepsin enzyme to tube 8. Rinse the cylinder 4. Add 2 ml of amylase enzyme to tube 9. Rinse the cylinder. 5. Add 2 ml of distilled water to tube 10. 6. Record the contents of all tubes in your data chart. 7. Put all test tubes in test tube rack in front of room for incubation at body temperature.
Day 2: 1. Set up a boiling water bath. 2. Get your test tubes from the test tube rack in the front of the room. 3. Add 10 drops of Benedict’s solution to each tube,. 4. Place all tubes in a boiling water bath and observe. 5. In you data table, note the number of the tube that shows a color change to yellow orange first. This color change indicates the presence of simple sugars. 6. As soon as the first tube changes color, unplug the water bath. Disregard further color changes

Answer the following questions on your paper:

5. Benedict’s reagent is use to test for the presence of simple sugars. Which test tube showed the presence of simple sugars? 6. Why does a positive test for sugar indicate starch digestion? 7. What do your results indicate about substrate specificity?

CONCLUSION:

On separate paper, type or write a conclusion summarizing the factors necessary for optimum enzyme action. The conclusion should begin with a brief introductory paragraph. Your first sentence should be an overall statement naming the four factors investigated in the lab, followed by a brief explanation of how enzymes work. The introductory paragraph should be followed by four paragraphs explaining what you learned about each aspect of enzyme action. Start each of the paragraphs with an overall statement about that aspect of enzyme action, then support the statement with data from the lab. DO NOT restate procedures in your conclusion. A final summary paragraph is not necessary. It is not necessary to discuss sources of error in this lab.

Four Factors Affecting

Enzyme Activity

1. pH Specificity

Enzyme Substrate Source of Enzyme

Contains pH Amount of Reaction Test tube #1

Test tube #2

Test tube #3

1. Based on your data, at what pH does catalase enzyme work best?

2. What effect does too much acid or too much base have on an enzyme? Be specific.

2. Temperature

Enzyme Substrate Source of enzyme

Contains Amount of Reaction Test tube #4

Test tube #5

1. Based on your data, what effect does boiling have on the effectiveness of catalase? Explain how you know.

2. What effect do you think very low temperatures (frozen raw liver) will have on the catalase enzyme?

3. At what approximate temperature does catalase enzyme work best? What effect does extreme temperatures have on the catalase?

3. Surface Area and Enzyme Action

Enzyme Substrate Source of Enzyme

Contains Amount of reaction

Test tube #6

Test tube #7

1. Which form of potato, raw, or mashes had more surface area to make catalase enzyme more available?

2. Which form of the potato showed more enzyme action-taking place? Why?

3. Substrate Specificity

Enzyme Substrate Source of Enzyme

Contains Amount of reaction

Test tube #8

Test tube #9

Test tube #10

1. What do the results indicate about substrate specificity?