AP Enzyme LAB2 S.pdf

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structure, either a pleated sheet or an α-helix, forms from hydrogen bonding interactions between polar side chains on the amino acids; tertiary structure results from the formation of disulfide bridges between amino acids containing sulfur atoms; and quaternary structure forms when more than one tertiary subunit binds together to make a functional protein. At any point in the secondary, tertiary, or quaternary structure, other chemicals in the solution may interact with the amino acids and change the conformation of the protein. When this occurs, the active site shape is changed, the protein is said to be denatured, and its catalytic ability is lost.

Increasing temperature in a solution is a measure of the increasing molecular motion of the molecules in that solution. Heating a solution in which an enzyme-catalyzed reaction is occurring may cause the reaction rate to increase. However, above a certain temperature (different for each type of enzyme), most enzymes will begin to lose their characteristic shape as the secondary, tertiary and quaternary interactions are disrupted. This results in a drastic decrease in the rate of the reaction as more and more enzyme molecules are denatured.

Recall that the measure of the concentration of free hydrogen ions (H+) in a solution is the pH of the solution. pH is calculated using a logarithmic scale that ranges from zero to 14. A low pH indicates a very acidic solution, or a high concentration of free H+in the solution. A high pH indicates a very alkaline, or basic, solution with a low concentration of free H+. Since all amino acids contain both a carboxyl group (-COOH) and an amino group (-NH2), every protein can act as either a H+acceptor if

the pH is low or a H+donor if the pH is high. At either extreme, the loss or gain of H+from the enzyme can cause a change in conformation that results in the decrease of enzyme activity.

Salts are formed by electrical attractions between ions with opposite charges. The resulting ionic bonds break when the salt is dissolved in polar solvents such as water,

very low salt concentration in a solution containing an enzyme may alter the enzyme’s conformation by

interacting with the charged side groups of the enzyme’s amino acids. Very low salt concentrations cause the amino acid side chains to form extra bonds with each. Very high salt concentrations disrupt some of the bonds between amino acids that would normally occur. Both of these situations can cause the enzyme to become denatured.

Each type of enzyme has an optimum temperature, pH, and salt concentration. Many enzymes found in the human body have an optimum temperature of around 37ºC (body temperature), an optimum pH of around 7 (neutral), and an optimum salt concentration of around 0.9% (the salt concentration of blood plasma and interstitial fluid). However, some enzymes function optimally under very different conditions, depending upon their specific function within the organism. For example, the protein-digesting enzyme found in the stomach, called pepsin, has an optimal pH of around 2. You will not be determining the optimum conditions for an enzyme in this laboratory. Instead you will be

determining the changes in the rate of the enzymatic reaction over a period of time.

The enzyme being investigated in the following

exercises, catalase, is a common enzyme found in nearly all living cells. It serves to detoxify some of the harmful chemicals produced during cellular metabolism. One such chemical, hydrogen peroxide (H2O2), is broken

down by catalase into two harmless products: water and oxygen gas. The summary equation for this reaction is:

Catalase structure varies somewhat between organisms. Bovine catalase, commonly used in the laboratory, has a molecular weight of 250,000 Daltons and is composed of four polypeptide subunits, each of approximately equal size. Bovine catalase functions best in an environment of

2 H2O2

➞

2 H2O + O2

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The rate of an enzymatic reaction can be determined if certain measurements are taken during the time that the enzyme is catalyzing the reaction. For example, if the amount of product produced by the reaction is

measured over a period of time, the reaction rate for that interval can be calculated. Likewise, if the amount of substrate present in the solution is measured over time, the reaction rate can be determined. If an enzyme and substrate are present in a solution, but the number of substrate molecules greatly outnumbers the enzyme molecules, then virtually all of the enzyme molecules will be actively catalyzing the reaction for some initial period of time. This is called the initial rate of the reaction, and is linear when graphed; the greater the slope of the line, the greater the initial rate of the reaction. This linear section of the graph is called the reaction rate, and is characteristic for a particular enzyme (given the same environmental conditions). Eventually, as the substrate molecules are used up in the formation of product, the number of enzyme molecules becomes greater than the number of substrate molecules. At this point, the graph changes from linear to a curve with a slope approaching zero. This indicates that the reaction rate is slowing down over that period of time.

As an example, use the graph below to determine the initial rate of an enzymatic reaction. To do this, pick two points in a linear on the area of the graph. Determine the difference in the amount of product present at these two points in time. This is the change in the Y-axis, and is represented by the symbol Δy. Now determine the amount of time that lapsed during this interval. This is the change in the X-axis, and is represented by the symbol Δx. Dividing the amount of product produced during this interval by the time during the interval gives you the rate of the reaction. Practice determining the rate of the reaction by filling in the table and completing the calculations below the sample graph.

0 10

5 15 20 25 30

P

roduc

t (mmol)

Time (s)

60 120 180 240 300 360 420 480

Reaction Rate Curve for an Enzyme-Catalyzed Reaction

Reaction ₌ ΔProduct(mmol) ₌ Δy ₌ mmol

Rate ΔTime (sec) Δx sec

Time (sec):

Product (mmol):

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This laboratory demonstrates the action of the enzyme catalase on hydrogen peroxide (H2O2). Hydrogen

peroxide is decomposed into water and oxygen gas by catalase activity. The amount of hydrogen peroxide that remains following various periods of catalase action will be measured to determine the rate of catalase activity.

• Observe

the effect of the addition of catalase to a

hydrogen peroxide solution.

• Predict

the effects of varying pH, temperature, substrate

concentration, and enzyme concentration on enzyme

reaction rates.

• Perform

titrations to determine the amount of H

2

O

2

remaining in various samples.

• Graph

the reaction rates of the enzyme catalase over a

period of time.

Part 1: THE UNCATALYZED RATE OF H

2 O

2 DECOMPOSITION

Day 1:

Per student Apron Gloves Goggles

Per group

30 mL 1.5% H2O2

1 Medicine cup

Marking pen or pencil Labeling tape

Be sure to always wear safety goggles, gloves and a lab apron to protect your eyes and clothing when working with any chemicals.

Dispose of any waste materials and clean up your work area as directed by your teacher.

Be sure to always wash your hands before leaving the laboratory.

Objectives

Activity 2a

The Uncatalyzed Rate of H

₂

O

₂

Decomposition

What You Need

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Step 1

Place 30 mL of 1.5% H2O2solution in a medicine cup

labeled “H2O2.”

Step 2

Set the uncovered cup aside for one week (168 hours) in an area where it will not be disturbed. This area should be at approximately room temperature.

Day 7:

Per group

Medicine cup with 1.5% H2O2(from Day 1)

2 Medicine cups 1 12-mL syringe, plastic 1 1-mL pipet

1 mL Distilled water 10 mL 1.0 M H2SO4

10 mL 2% KMnO4

Paper, white

Sulfuric acid (H2SO4) is a strong acid and can be a caustic

irritant if allowed to come in contact with the skin. In case of spills or skin contact, inform your teacher immediately and flush areas with running water for 15 minutes.

Potassium permanganate (KMnO4) will stain skin,

equipment, and clothing if contact occurs. Be sure to wear appropriate laboratory protective clothing and observe proper laboratory techniques to avoid contact with KMnO4.

Step 1

Remove 10 mL of the H2O2from the cup using the 12

mL syringe, and transfer it to another medicine cup labeled “Uncatalyzed H2O2Activity.”Thoroughly rinse

the syringe after using it.

Step 2

Using the 1 mL pipet, add 1 mL of distilled water to the H2O2in the Uncatalyzed H2O2Activity cup.

What to do. . .

What You Need

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Step 3

Using the 12 mL syringe, add 10 mL of 1.0 M H2SO4to

the cup. Thoroughly rinse the syringe after using it.

CAUTION:Sulfuric acid (H2SO4) is a strong acid and can

cause chemical burns. Use extreme caution when handling any acids. If spills or skin contact occur, inform your teacher immediately and flush the affected skin with running water for 15 minutes.

Step 4

Swirl gently to mix the contents of the cup.

Step 5

Label the remaining medicine cup “Uncatalyzed Assay.” Using the 12 mL syringe, transfer 5 mL of the

Uncatalyzed H2O2Activity mixture to this cup.

Thoroughly rinse the syringe after using it.

Step 6

Draw 10 mL of 2% KMnO4into the 12 mL syringe.

CAUTION:Potassium permanganate (KMnO₄) will stain

clothes, skin, and equipment. Use care when handling it.

Step 7

Place a white sheet of paper on the counter where you will be performing the titration. Place the Uncatalyzed Assay cup on the white sheet of paper. Read the initial volume of KMnO4in the syringe and record this in Data

Table 1.

Step 8

Holding the 12 mL syringe over the cup, carefully

depress the plunger so that one drop of KMnO4falls into

the cup. Swirl the cup gently and observe the solution for any pink to brown coloration that remains after mix-ing is complete.

Step 9

Complete the titration by repeating Step 8, drop by drop, until a pink to brown coloration of the solution persists after mixing. Record the final volume of the KMnO4

solu-tion in the 12 mL syringe in Data Table 1.

Step 10

Be sure to wash your hands and clean up and dispose of any waste materials as directed by your teacher.

Analysis

Data Table 1: The Uncatalyzed Rate of H₂O₂Decomposition

Initial KMnO4volume in 12 mL syringe (mL):

Final KMnO4volume in 12 mL syringe (mL):

KMnO4Used in Uncatalyzed Titration

(Initial – Final):

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Part 2: TEST OF CATALASE ACTIVITY

Per group

30 mL1.5% H2O2

3 Medicine cups

6 mL Catalase solution in medicine cup 1 Test tube

1 Test tube holder 1 Wood macerating stick

1 cm2_{cube of liver and/or potato}

12 mL Syringe 1 mL Pipet

Shared Materials Hot plate

250 mL Beaker with water for boiling catalase solution Container with ice for catalase solution

Test tube rack

Knife or scalpel for cutting cubes of potato and/or liver

Use caution when using sharp instruments in the laboratory.

Part A: Observation of Catalase Action

Step 1

Using the 12 mL syringe, place 10 mL of 1.5% H2O2into

a medicine cup labeled “Catalase Extract Activity.” Thoroughly rinse the syringe after using it.

Step 2

Using the 1 mL pipet, add 1 mL catalase solution to the H2O2in the medicine cup. Be sure to keep the remainder

of the catalase solution in the ice container whenever it is not being used. Thoroughly rinse the pipet after using it.

Step 3

Gently swirl the cup to mix the contents, and observe the reaction occurring in the cup. Record your observa-tions in Data Table 2.

What You Need

Safety

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Part B: The Effect of Boiling on Catalase Action

Step 1

Using the 12 mL syringe, place 5 mL catalase solution into a test tube. Thoroughly rinse the syringe after using it.

Step 2

Using the test tube holder, place the test tube containing the catalase solution into the boiling water bath.

Step 3

After 5 minutes, remove the tube from the boiling water bath with the test tube holder, and place it in the test tube rack to cool.

Step 4

While the catalase solution is cooling, place 10 mL of the 1.5% H2O2solution in a medicine cup labeled “Boiled

Catalase Activity.”Thoroughly rinse the syringe after using it.

Step 5

When the catalase solution in the test tube has cooled to room temperature, add 1 mL to the H2O2in the

medicine cup.

Step 6

Gently swirl the cup to mix the contents, and observe any reaction occurring in the cup. Record your observations in Data Table 2.

Part C: Catalase Action in Living Tissue

Step 1

Obtain a 1 cm2_{cube of liver or potato tissue, and place it}

in a medicine cup labeled “Live Tissue Catalase Activity.”

Step 2:

Use the end of the wood macerating stick to grind the tissue into small pieces.

Step 3:

Add 10 mL of the 1.5% H2O2solution to the macerated

tissue in the cup. Gently swirl the cup to mix the contents, and observe any reaction taking place in the cup. Record your observations in Data Table 2.

Thoroughly rinse the syringe after using it.

Step 4:

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Analysis

Write the overall reaction for this enzymatic reaction:

Data Table 2: Results of Catalase Activity, Effect of Boiling on Catalase, and Catalase Activity in Living Tissue

Experiment: Substrate

Test of Catalase Activity (Part A)

Effect of Boiling on Catalase (Part B)

Catalase from Living Tissue (Part C)

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Part 3: Baseline Assay and Enzyme-catalyzed

Rate of H

₂

O

₂

Decomposition

Per group

70 mL 1.5% H2O2in 120 mL graduated cup

8 Medicine cups 1 6-mL syringe 1 1-mL pipet

1 mL Distilled water in medicine cup 6 mL Catalase solution in medicine cup 70 mL 1.0 M H2SO4in glass beaker

40 mL 2% KMnO4in 120 mL graduated cup

1 12-mL Syringe, plastic Marking pen or pencil Labeling tape

Stopwatch or clock with second hand Paper, white

Shared Materials

Container with ice for catalase solution

Sulfuric acid (H2SO4) is a strong acid and can be a caustic

irritant if allowed to come in contact with the skin.

In case of spills or skin contact, inform your teacher immediately and flush areas with running water for 15 minutes.

Potassium permanganate (KMnO4) will stain skin,

equipment, and clothing if contact occurs. Be sure to wear appropriate laboratory protective clothing and observe proper laboratory techniques to avoid contact with KMnO4.

Step 1

Label seven of the eight medicine cups as shown in the table below. For cups 1-6, the label indicates the length of time that the reaction will be allowed to proceed.

What You Need

Safety

What to do. . .

1 10 seconds

2 30 seconds

3 60 seconds

4 120 seconds

5 180 seconds

6 360 seconds

7 Baseline

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Step 8

Repeat Steps 6 and 7 with Cups 2-6, timing each for the length indicated on their labels. Set each aside for titra-tion after the reactitra-tion is completed. Thoroughly rinse the pipet and syringe after completing this step.

Step 9

Label the remaining medicine cup “Titration Vessel.” Using the 6 mL syringe, transfer 5 mL of the solution from Cup 7 (Baseline) to the titration vessel. Save the remainder of each solution until all data are collected. Thoroughly rinse the syringe after using it.

Step 10

Draw 12 mL of 2% KMnO4into the 12 mL syringe.

CAUTION:Potassium permanganate (KMnO4) will stain

clothes, skin, and equipment. Use care when handling it.

Step 11

Place a white sheet of paper on the counter where you will be performing the titration. Place the titration vessel on the white sheet of paper. Read the initial volume of KMnO4in the syringe and record this in Data Table 3.

Step 12

Holding the syringe over the titration vessel, carefully depress the plunger so that one drop of KMnO4falls into

the cup. Swirl the cup gently and observe the solution for any pink to brown coloration that remains after mix-ing is complete.

Step 13

Complete the titration by repeating Step 12, drop by drop, until a pink to brown coloration of the solution persists after mixing. Record the final volume of the KMnO4solution in the 12 mL syringe in Data Table 3.

Step 2

Into each of the seven labeled cups, add 10 mL of 1.5% H2O2solution. Thoroughly rinse the 12 mL syringe after

completing this step.

Step 3

Using the 1 mL pipet, add 1 mL of distilled water to the H2O2in Cup 7 (Baseline).

Step 4

Using the 12 mL syringe, add 10 mL of 1.0 M H2SO4to

Cup 7 (Baseline).CAUTION:Sulfuric acid (H2SO4) is a strong acid and can cause chemical burns. Use extreme caution when handling any acids. If spills or skin contact occur, inform your teacher immediately and flush the affected skin with running water for 15 minutes.

Step 5

Swirl gently to mix the contents of Cup 7. Set this cup aside for later titration.

Step 6

Have the stopwatch or clock with second hand ready to begin timing the reaction. Refill the 12 mL syringe with 10 mL of 1.0 M H2SO4and have it ready to add to the

reaction mixture.

Step 7

Using the 1 mL pipet, add 1 mL of catalase extract to Cup 1 and begin timing (10 seconds). Swirl the mixture gen-tly. At the end of 10 seconds, add the 10 mL of H2SO4

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Step 14

Dispose of the sample in the titration vessel according to instructions from your teacher. Rinse and dry the titration vessel. Repeat the titration on 5 mL of each of the remaining reaction mixtures (10, 30, 60, 120, 180, 360 seconds). Be sure to clean and dry the titration vessel in between each titration. Record your data for each mixture in Data Table 4.

Step 15

Be sure to wash your hands and clean up and dispose of any waste materials as directed by your teacher.

Step 16

Perform the calculations for H2O2decomposition and

catalase reaction rates in Data Tables 3, 4, and 5. The reaction rate data from Data Table 5 will then be used to construct a graph.

NOTE:The volume (mL) of KMnO₄used in each activity in

this laboratory is proportional in a 1:1 ratio to the volume (mL) of H2O2decomposed.

Analysis

1. Initial KMnO4 volume in 12 mL syringe (mL): 2. Final KMnO4 volume in 12 mL syringe (mL): 3. KMnO4 Used in Baseline Titration

(Initial – Final, mL):

4. Baseline mL H2O2 present in 5 mL sample:

Data Table 3: Calculation of Baseline mL H2O2and %

Spontaneous Decomposition of H2O2in 24 hours

Baseline Assay

Calculation of Spontaneously Decomposed H₂O₂ 5. KMnO4 used in Uncatalyzed Reaction (from

Data Table 1):

6. Baseline mL KMnO4 (Line 3) – mL of KMnO4 Used in Uncatalyzed Titration (Line 5): 7. Volume (mL) of spontaneously decomposed

H2O2:

8. % Spontaneous Decomposition of H2O2 in 24 hours:

(mL Baseline – mL Uncatalyzed)/7 mL Baseline 100

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Calculate the rate of the reaction for each of the time increments listed in Data Table 5 by dividing the difference in volume of H2O2in one time interval by the number of seconds in that interval. This is the same calculation necessary

to determine the slope of a line:

________ y

________ x 1. Baseline KMnO₄Used

(from Data Table 3): Note: Enter this value in all

columns in this row.

2. Initial KMnO₄volume in 12 mL syringe:

3. Final KMnO₄volume in 12 mL syringe:

4. KMnO4 used in each timed titration (Initial – Final):

Data Table 4: Results of Timed Catalase Reaction

KMnO4Volumes (mL):

5. Volume of H2O2decomposed at each time interval (line 1- line 4)

Time (s)

10 30 60 120 180 360

Calculation of H2O2(mL) Decomposition

Data Table 5: Reaction Rates for Catalase Decomposition of H₂O₂

Rate of Reaction: (mL H₂O₂/sec)

Time Increments

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Graphing the results of the enzyme-catalyzed

decom-position of H₂O₂:

You will construct a graph of the rate of catalase action below. Use the following instructions to construct the graph: Plot the dependent variable on the Y-axis; plot the independent variable on the X-axis. Don’t forget to label each axis with a descriptive label as well as any appropri-ate units. Give the graph an appropriappropri-ate title as well.

The independent variablein a scientific experiment is

the condition that the experimenter deliberately varies. What condition in this experiment did you allow to vary to see if that had any effect on the experiment’s results?

Independent variable:

________________________________

The dependent variablein a scientific experiment is

what the experimenter designed the experiment to measure. What variable did you measure at the comple-tion of this experiment?

Dependent variable:

________________________________

0 0 2

1 3 4 5

1.5

0.5 2.5 3.5 4.5

60 120 180 240 300 360

H2 O2

(mL) D

e

co

mposed

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Questions

1. Why was performing a baseline assay necessary to interpret the results of your experiments? (Hint:What information did the baseline assay provide you with?)

...

2. Why was performing an assay on uncatalyzed H2O2

important? What does this information tell you about the shelf life of H2O2, which is sometimes used as a

disinfectant?

...

3. Explain the results of the exercise that used boiled catalase extract. What would your predictions have been for an experiment using cooked liver instead of fresh liver as a catalase source?

...

4. Why was H2SO4used in these experiments? How did

adding H2SO4have the desired effect on the reaction

mixture?

...

5. During which time increment on your graph is the reaction rate (the slope of the line) the steepest? Why?

...

6. During which time increment(s) on your graph is the reaction rate the slowest? Why?

...