AP Lab 2 Report 2001

 

Enzyme Catalysis

 

Introduction
Enzymes are proteins produced by living cells that act as catalysts, which affect the rate of a biochemical reaction. They allow these complex biochemical reactions to occur at a relatively low temperature and with less energy usage.

In enzyme-catalyzed reactions, a substrate, the substance to be acted upon, binds to the active site on an enzyme to form the desired product. Each active site on the enzyme is unique to the substrate it will bind with causing each to have an individual three-dimensional structure. This reaction is reversible and is shown as following:

E + S—-ES—- E + P

Enzymes are recyclable and unchanged during the reaction. The active site is the only part of the enzyme that reacts with the substrate. However, its unique protein structure under certain circumstances can easily be denatured. Some of the factors that affect enzyme reactions are salt concentration, pH, temperature, substrate and product concentration, and activators and inhibitors.

Enzymes require a very specific environment to be affective. Salt concentration must be in an intermediate concentration. If the salt concentration is too low, the enzyme side chains will attract each other and form an inactive precipitate. Likewise, if the salt concentration is too high, the enzyme reaction is blocked by the salt ions. The optimum pH for an enzyme-catalyzed reaction is neutral (7 on the pH scale). If the pH rises and becomes basic, the enzyme begins losing its H+ ions, and if it becomes too acidic, the enzyme gains H+ ions. Both of these conditions denature the enzyme and cause its active site to change shape.

Enzymes also have a temperature optimum, which is obtained when the enzyme is working at its fastest, and if raised any further, the enzyme would denature. For substrate and product concentrations, enzymes follow the law of mass action, which says that the direction of a reaction is directly dependent on the concentration. Activators make active sites better fit a substrate causing the reaction rate to increase. Inhibitors bind with the enzymes’ active site and block the substrate from bonding causing the reaction to subside.

The enzyme in this lab is catalase, which produced by living organisms to prevent the accumulation of toxic hydrogen peroxide. Hydrogen peroxide decomposes to form water and oxygen as in the following equation:

2H2O2 ® 2H2O + O2

This reaction occurs spontaneously without catalase, but the enzyme speeds the reaction considerably. This lab’s purpose is to prove that catalase does speed the decomposition of hydrogen peroxide and to determine the rate of this reaction.

 

Hypothesis
The enzyme catalase, under optimum conditions, effectively speeds the decomposition of hydrogen peroxide.

 

Materials
Exercise 2A: Test of Catalase Activity

In Part 1, the materials used were 10mL of 1.5% H2O2, 50-mL glass beaker, 1 mL catalase, and 2 10-mL pipettes and pipette pumps. In Part 2, the materials used were 5 mL of catalase, a boiling water bath, 1 test tube, a test tube rack, 10 mL of 1.5% H2O2, 50-mL beaker, and 2 10-mL pipettes and pipette pumps. In Part 3, the materials used were 10 mL of 1.5% H2O2, 50-mL beaker, liver, and a syringe.

Exercise 2B: The Baseline Assay

This part of the lab required 10 mL of 1.5% H2O2, 1 mL distilled H2O, 10 mL of H2SO4, 2 50-mL beakers, a sheet of white paper, 5 mL KMnO4, 2 5-mL syringes, and 2 10-mL pipettes and pumps.

Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition

The materials used for this section were 15 mL of 1.5% H2O2, 1 mL distilled H2O, 10 mL H2SO4, 2 50-mL beakers, a sheet of white paper, 5 mL KMnO4, 2 5-mL syringes, and 2 10-mL pipettes and pumps.

Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition

The materials required for Exercise 2D were 70 mL of 1.5% H2O2, 70 mL of H2SO4, 6 mL of catalase solution, 13 plastic, labeled cups, 3 100-mL beakers, 1 50-mL beaker, 1 10-mL syringe, 1 5-mL syringe, 1 60-mL syringe, a sheet of white paper, a timer, and 30 mL of KMnO4.

 

Method
Exercise 2A: Test of Catalase Activity

In Part 1, 10 mL of 1.5% H2O2 were transferred into a 50-mL beaker. Then, 1 mL of fresh catalase solution was added and the reaction was observed and recorded. In Part 2, 5 mL of catalase was placed in a test tube and put in a boiling water bath for five minutes. 10 mL of 1.5% H2O2 were transferred to a 50-mL beaker and 1 mL of the boiled catalase was added. The reaction was observed and recorded. In Part 3, 10mL of 1.5% H2O2 were transferred to a 50 mL beaker. 1 cm3 of liver was added to the beaker and the reaction was observed and recorded.

Exercise 2B: The Baseline Assay

10 mL of 1.5% H2O2 were transferred to a 50-mL beaker. 1 mL of H2O was added instead of catalase, and then, 10 mL of H2SO4 were added. After mixing well, a 5 mL sample was removed and placed over a white sheet of paper. A 5-mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded. The baseline assay was calculated.

Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition

A small quantity of H2O2 was placed in a beaker and stored uncovered for approximately 24 hours. To determine the amount of H2O2 remaining, 10 mL of 1.5% H2O2 were transferred to a 50-mL beaker. 1 mL of H2O was added instead of catalase, and then, 10 mL of H2SO4 were added. After mixing well, a 5 mL sample was removed and placed over a white sheet of paper. A 5-mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded. The percent of the spontaneously decomposed H2O2 was calculated.

Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition

 

The baseline assay was reestablished following the directions of Exercise 2B. Before starting the actual experiment a lot of preparation was required. Six labeled cups were set out according to their times and 10 mL of H2O2 were added to each cup. 6 mL of catalase were placed in a 10-mL syringe, and 60 mL of H2SO4 were placed in a 60-mL syringe. To start the actual lab, 1 mL of catalase was added to each of the cups, while simultaneously, the timer was started. Each of the cups were swirled. At 10 seconds, 10 mL of H2SO4 were added to stop the reaction. The same steps were repeated for the 30, 60, 120, 180, and 360 second cups, respectively.

Afterwards, a five 5 mL sample of each of the larger cups were moved to the corresponding labeled smaller cups. Each sample was assayed separately by placing each over a white sheet of paper. A 5-mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded.

 

Results

Table 1
Enzyme Activity

 

 

 

Activity

 

Observations

Enzyme activity The solution only bubbled slightly and slowly.
Effect of Extreme temperature

 

 

The catalase had no reaction with the H2O2; there were no bubbles
Presence of catalase The solution foamed up immediately

 

 

Table 2
Establishing a Baseline

 

 

 

Volume

 

Initial reading

 

 

5.0 mL

 

Final reading

 

 

0.8 mL

 

Baseline ( final volume – initial volume)

 

 

4.2 mL

 

 

Table 3
Rate of Hydrogen Peroxide Spontaneous Decomposition

 

 

 

Volume

 

Initial KMnO4

 

 

5.0 mL

 

Final KMnO4

 

 

1.2 mL

 

Amount of KMnO4 used after 24 hours

 

 

3.8 mL

 

Amount of H2O2 spontaneously decomposed
( ml baseline – ml after 24 hours)

 

0.4 mL

 

Percent of H2O2 spontaneously decomposed
( ml baseline – ml after 24 hours/ baseline)

 

9.52%

 

 

Table 4
Rate of Hydrogen Peroxide Decomposition by Catalase

 

Time ( Seconds)
10 30 60 120 180 360
 

Baseline KMnO4

 

 

4.0 mL

 

4.0 mL

 

4.0 mL

 

4.0 mL

 

4.0 mL

 

4.0 mL

 

Initial volume KMnO4

 

 

5.0 mL

 

5.0 mL

 

5.0 mL

 

5.0 mL

 

5.0 mL

 

5.0 mL

 

Final volume KMnO4

 

 

2.2 mL

 

1.4 mL

 

2.0 mL

 

1.7 mL

 

2.4 mL

 

2.3 mL

 

Amount KMnO4 used
(baseline – final)

 

2.8 mL

 

3.6 mL

 

3.0 mL

 

3.3 mL

 

2.6 mL

 

2.7 mL

 

Amount H2O2 used
(KMnO4 – initial)

 

1.2 mL

 

0.4 mL

 

1.0 mL

 

0.7 mL

 

1.4 mL

 

1.3 mL

 

Amount of Hydrogen Peroxide Decomposed by Catalase

Exercise 2A: Test of Catalase Activity

1. Observing the reaction of catalase on hydrogen peroxide:

a. What is the enzyme in this reaction?  catalase

b. What is the substrate in this reaction? Hydrogen peroxide

c. What is the product in this reaction? Oxygen & water

d. How could you show that the gas evolved is O2? The gas could be shown to be O2 if the gas were collected in a tube, and a glowing splint was held in the tube. If the splint glowed, it would prove the gas was oxygen.

2. Demonstrating the effect of boiling on enzyme action:

a. How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference. While the unboiled catalase caused bubbles to form in the solution, the boiled catalase did not react at all because boiling an enzyme causes the protein to unfold and therefore denatures it.

3. Demonstrating the presence of catalase in living tissue:

a. What do you think would happen if the potato or liver was boiled before being added to the H2O2? The catalase in the liver would have been denatured by the boiling and would not have reacted with the H2O2.

Analysis of Results

1. Determine the initial rate of the reaction and the rates between each of the time points.

 

 

Time Intervals (Seconds)

 

Initial 0 to 10

 

10 to 30

 

30 to 60

 

60 to 120

 

120 to 180

 

180 to 360

 

Rates

 

0.12 mL/sec

 

-0.04 mL/sec

 

0.02 mL/sec

 

-0.005 mL/sec

 

0.01167 mL/sec

 

-0.00083

mL/sec

 

 

2. When is the rate the highest? Explain why.

 

The rate is the highest in the initial ten seconds because the concentration of catalase is at its highest. As more of the product is formed, it blocks the reaction between the catalase and the hydrogen peroxide.

3. When is the rate the lowest? For what reasons is the rate low?

The rate is lowest during the 180-360 seconds time period because of the law of mass action. This law says that when there is a high concentration of product as in this period, the enzymes will be blocked by the product (water) from reaching and reacting with the substrate (H2O2).

 

4. Explain the inhibiting effect of sulfuric acid on the function of catalase. Relate this to enzyme structure and chemistry

 

Sulfuric acid has an inhibiting effect on catalase function because it causes the pH level in the solution to lower considerably. Acidic solutions cause the protein structure of the enzyme to gain H+ ions causing it to denature.

 

5. Predict the effect lowering the temperature would have on the rate of enzyme activity. Explain your prediction.

 

Lowering the temperature of the catalase would slow the rate of reaction until it finally caused the enzyme to denature, and it would no longer react with the substrate. Most enzymes are only affective in a temperature range between 40° – 50° C.

6. Design a controlled experiment to test the effect of varying pH, temperature, or enzyme concentration.

Part 1: Enzyme Activity at Room Temperature

Add 10 mL of 1.5% H2O2 to a 50-mL beaker, and add 1 mL of room temperature catalase. Mix well and add 10 mL of H2SO4. Watch the reaction and record the results.

Part 2: The Effect of Excessive Heat on Enzyme Activity

Put 5 mL of catalase into a test tube and heat thoroughly over a Bunsen burner. Add 1 mL of the heated catalase to 10 mL of 1.5% H2O2 in a 50-mL beaker. Add 10 mL of H2SO4. Watch the reaction and record the results.

Part 3: The Effect of Excessive Cooling on Enzyme Activity

Put 5 mL of catalase in a freezer until completely frozen. Add 1 mL of the frozen catalase to 10 mL of 1.5% H2O2 in a 50-mL beaker. Add 10 mL of H2SO4. Watch the reaction and record the results.

 

Error Analysis
Any number of factors in this lab could have affected the results of this experiment. To get the desired results all of the measurements had to be precisely accurate and fully planned before hand. In Exercise D especially, the factor of planning became increasingly essential. The first attempt at 2D was unsuccessful due to several reasons. First of all, the measurements, which were taken, could have possibly been inaccurate and the 60-mL syringe containing H2SO4 also dripped into one of the cups early which did not allow the reaction to fully take place. There was also some confusion on the operation of the timer and precise planning in its use. The second attempt at 2D contained errors as well. The measurements were still not as accurate as they should have been, and the solution did not appear entirely uniform. In one cup, for example, the first drop of KMnO4 left a persistent pink color, and then after over a minute, it returned back to being clear. It then took several milliliters more to get it back to a pink color.

 

Discussion and Conclusion
This lab showed how catalase increased the rate of decomposition of hydrogen peroxide. In 2A, it was shown that catalase causes a visual reaction with H2O2, that when boiled catalase is no longer reactive, and that catalase is present in living tissue. Lab 2C shows that the natural decomposition of H2O2 is much slower than the enzymatic reaction. Lab 2D showed the decomposition of H2O2 over just a period of six minutes, and it had already decomposed more than the uncatalyzed H2O2 had done in 24 hours.

BACK

 

Chemistry Puzzle

 

Chemistry

Across
2. solution with an excess of hydronium (H+) ions
4. universal solvent
8. substances in organism that control pH
9. reactions involving a net release of energy
12. made of 2 or more elements bonded together
14. chemically combining elements so their atoms become stable
17. found in the energy levels of an atom
19. positive particle in the nucleus
20. quantity of matter in an object
21. substance dissolved in a solution
22. smallest part of a compound
24. center of an atom
Down
1. solution in which no more solute may be dissolved
2. refers to a basic solution
3. symbol for helium
5. neutral particle in the nucleus
6. solution in which water is the solvent
7. found on the left side of an equation
10. hydroxide ion
11. reaction involving a net absorption of energy
13. scale from 0-14 measuring H+ and OH- ion concentration
15. charged atom
16. reactions involving the transfer of electrons between atoms
18. pure substances that cannot be chemically broken down into simpler substances
22. anything that occupies space & has mass
23. stored in chemical bonds

CLICK HERE FOR NOTEBOOK COPY

Properties of Water

 

Properties of Water

 

Introduction:

Water’s chemical description is H2O. As the diagram to the left shows, that is one atom of oxygen bound to two atoms of hydrogen. The hydrogen atoms are “attached” to one side of the oxygen atom, resulting in a water molecule having a positive charge on the side where the hydrogen atoms are and a negative charge on the other side, where the oxygen atom is. This uneven distribution of charge is called polarity. Since opposite electrical charges attract, water molecules tend to attract each other, making water kind of “sticky.” As the right-side diagram shows, the side with the hydrogen atoms (positive charge) attracts the oxygen side (negative charge) of a different water molecule. (If the water molecule here looks familiar, remember that everyone’s favorite mouse is mostly water, too). This property of water is known as cohesion.

All these water molecules attracting each other mean they tend to clump together. This is why water drops are, in fact, drops! If it wasn’t for some of Earth’s forces, such as gravity, a drop of water would be ball shaped — a perfect sphere. Even if it doesn’t form a perfect sphere on Earth, we should be happy water is sticky. Water is called the “universal solvent” because it dissolves more substances than any other liquid. This means that wherever water goes, either through the ground or through our bodies, it takes along valuable chemicals, minerals, and nutrients.

Water, the liquid commonly used for cleaning, has a property called surface tension. In the body of the water, each molecule is surrounded and attracted by other water molecules. However, at the surface, those molecules are surrounded by other water molecules only on the water side. A tension is created as the water molecules at the surface are pulled into the body of the water. This tension causes water to bead up on surfaces (glass, fabric), which slows wetting of the surface and inhibits the cleaning process. You can see surface tension at work by placing a drop of water onto a counter top. The drop will hold its shape and will not spread.

In the cleaning process, surface tension must be reduced so water can spread and wet surfaces. Chemicals that are able to do this effectively are called surface active agents, or surfactants. They are said to make water “wetter.” Surfactants perform other important functions in cleaning, such as loosening, emulsifying (dispersing in water) and holding soil in suspension until it can be rinsed away. Surfactants can also provide alkalinity, which is useful in removing acidic soils.

Pre-Lab Questions (Click here)

Materials:

Box of small paper clips, small plastic container, eyedropper, cup, stirring rod, water, liquid soap, plastic tray

Procedure (Part A) Cohesiveness of Water:

  1. Estimate how many paper clips will fit into a completely full cup of water. Record this number in data table 1.
  2. Place your small container on a tray to contain any water that may spill.
  3. Fill a plastic cup with tap water.
  4. Pour tap water from your cup into your small container.
  5. Continue to add water by eyedropper until the top surface appears rounded.
  6. Slowly add paper clips one at a time to the cup keeping count of all paper clips that you add.
  7. Stop adding paper clips to the container whenever water spills from the top.
  8. Record your paper clip count. Compare the actual number of paper clips to the estimated number.

Procedure (Part B) Soap’s effect on Surface Tension:

  1. Again estimate how many paper clips will fit into a completely full cup of soapy water. Record this number in data table 2.
  2. Place your small container on a tray to contain any water that may spill.
  3. Fill a plastic cup with tap water.
  4. Add several drops of liquid soap & use a stirring rod to mix.
  5. Pour soapy water from your cup into your small container.
  6. Continue to add soapy water by eyedropper until the top surface appears rounded.
  7. Slowly add paper clips one at a time to the cup keeping count of all paper clips that you add.
  8. Stop adding paper clips to the container whenever water spills from the top.
  9. Record your paper clip count. Compare the actual number of paper clips to the estimated number.

Data:

Table 1

 

Cohesiveness of Tapwater
Estimated Number of Paper Clips Actual Number of paper Clips Difference
 

 

 

Table 2

 

Cohesiveness of Soapy water
Estimated Number of Paper Clips Actual Number of paper Clips Difference
 

 

 

Questions: 

1. How did your estimated number compare to your actual number?

2. What happened to the surface of the water as more clips were added?

 

3. What property of water was shown in Part A?

4. How is this property of water used in nature?

5. Explain why water shows surface tension.

 

6. Explain why water is a polar molecule and include a diagram of several water molecules in a drop of water.

 

 

7. In order to clean a surface, what must happen to surface tension?

 

8. What is the job of a surfactant?

 

9. Name a surfactant used in Part B?

10. Using your data from Part B, explain what proof you gathered in Part B to support your answer to question 9.

 

 

 

pH in Living Systems

 

 

pH and Living Systems

 

Introduction:

Scientists use something called the pH scale to measure how acidic or basic a liquid is. The scale goes from 0 to 14. Distilled water is neutral and has a pH of 7. Acids are found between 0 and 7. Bases are from 7 to 14. Most of the liquids you find every day have a pH near 7. They are either a little below or a little above that mark. When you start looking at the pH of chemicals, the numbers go to the extremes. Substances with the highest pH (strong bases) and the lowest pH (strong acids) are very dangerous chemicals. Molecules that make up or are produced by living organisms usually only function within a narrow pH range (near neutral) and a narrow temperature range (body temperature). Many biological solutions, such as blood, have a pH near neutral.

The biological molecule used in this lab is a protein found in milk. Proteins are used to build cells and do most of the cell’s work. They also act as enzymes. For proteins to work, they must maintain their globular shape. Changing the shape of a protein denatures and the protein will no longer work.

Materials:

Small squares of wide-range pH paper, pH color chart, paper towels, 4 dropper bottles, ammonia, lemon juice, skim milk, distilled water, forceps, 50 ml beakers, small squares of narrow-range pH paper, 2 stirring rods

Procedure (part A): Testing the pH of Substances

  1. Line up 4 squares of wide-range pH paper about 1 cm apart on a paper towel.
  2. Put one drop of distilled water on the pH square.
  3. Compare the color of the pH paper to the color chart and record the pH in data table 1.
  4. Repeat this procedure for the ammonia, lemon juice, and skim milk.

Questions (Part A): Determining the pH of Solutions

  1. Which substance was the most acidic?
  2. Which substance was the most basic?
  3. Did any of the substances have a pH close to neutral? Name them.

Procedure (part B): Showing the Effect of pH on a Biological Molecule (Milk Proteins)

  1. Place 100 drops of skim milk in a 50 ml beaker.
  2. Pick up a piece of narrow-range pH paper with forceps.
  3. Touch the pH paper to the milk and remove it.
  4. Compare the color of the pH paper to the pH color chart.
  5. Record the initial pH in data table 2.
  6. Add a drop of lemon juice to the milk in the cup & stir with a stirring rod. Keep track of how many drops you add to the milk!
  7. Measure and record the pH of the solution with the narrow-range pH paper.
  8. Repeat step 7 until you notice an obvious change in the appearance of the milk. record this final pH and appearance of the milk in your data table.
  9. Repeat steps 1-8 using a clean 50 ml beaker and fresh milk, and substitute ammonia for the lemon juice.
  10. Add drops of ammonia to the milk until the change in pH of the milk is equal to the change in pH you measured in step 8. Be sure to keep track of the number of drops added. HINT: If the pH changed by 2 units with the lemon juice, then add ammonia until you also get 2 pH units of change!

Data:

Table 1

 

Substance Tested pH Acid Base Neutral

 

Table 2

Substance Tested Substance used to Produce Change Starting pH of Milk Final pH of Milk Original Appearance of Milk Final Appearance of Milk Total Number of drops added to Produce the change
100 drops Skim Milk Lemon Juice
100 drops Skim Milk Ammonia

Questions:

1. Which substance tested from table 1 was the most acidic?

2. Which substance was most basic?

3. Did any substance from table 1 have a neutral, or near neutral pH? If so, which substance was neutral?

4. Why did you use narrow-range pH paper to measure the milk’s change in pH?

 

5. Describe the change in appearance of the milk as more lemon juice was added. Explain why this change occurred.

 

 

6. How much did the pH of milk change when lemon juice was added?

7. Why do you think lemon juice “curdled”  (precipitated out the proteins) from the milk?

 

8. Did you get the same change when ammonia was used? Why or why not?