Category: Biochemistry of Organisms

 

Lab 2    Enzyme Catalysis

 

Introduction

The human body produces many things to keep it alive and healthy. Enzymes are proteins produced by living cells. Enzyme-catalysis binds with the active site of an enzyme, reducing the amount of energy needed to have a reaction with the substrate. Catalysis is a substance that lowers reaction energy and allows the reaction to take place in less time and at lower temperatures. Without catalysis people would die from poisons that the body produces, but would not be able to break down. Catalysis does not break down during a reaction so can be used reversibly (E is enzyme, S is substrate, and P is Product):

E+S↔ES↔E+P

Even with catalysis, enzyme reactions can be affected by many factors: salt concentration, pH, temperature, substrate concentration, enzyme concentration, activators, and inhibitors. Salt concentration affects the enzyme if it is to high or to low. Not enough salt will cause the enzyme side chains to become attracted to each other and denaturalizes it. Too much salt blocks the action site of the enzyme. The pH of a substance is determined by the amount of hydrogen (H+) in it. The scale ranges from 0-14: 0-7 is acidic, 7 is neutral, and 7-14 is basic. If the pH is too basic, the enzyme gains (H+) and eventually denaturalizes. If the pH is too acidic, then the enzyme loses (H+) and becomes denaturalized. The ideal pH is between 6 and 8. Temperature affects the kinetic energy that causes the reaction to speed up or slow down. In general, the higher the temperature the faster the molecular reaction. If the enzyme raises to a temperature above its optimum level, the tertiary structure of the protein is destroyed (denaturing it). Most enzymes denaturalize around 40-50°C. The law of mass action states the direction of enzyme-catalyzed reaction is dependent on conservation of enzyme/substrate/product. For example, when the substance has high substrate and low product, the substrate is used and more products are made. When the product is high and enzyme low, the reaction reverses and produces more substrate. If the product is immediately metabolized or removed from the cell, there will be no substrate formed. Activators increase the rate of a reaction while inhibitors slow the rate of the reaction. Inhibitors unfold an enzyme or reduce the –S-S- chains that stabilize the enzyme’s structure. Some enzyme inhibitors are poisons like potassium cyanide and curare.

Even without catalase, a reaction will still occur, but slower. The study of kinetics helps to determine the amount of product or substrate formed.

Chemical reactions occur over periods of time. The first three minutes of the reaction, the rate of change stays about the same. After a while, when there is less substrate, the reaction slows down and the rate of change becomes less. To compare the change of kinetic energy between reactions, a common point must be obtained. The first part of the reaction is called the initial rate of change. The initial rate of any enzyme-catalyzed reaction can be determined by the characteristics of the enzyme molecule. This is always the same for any enzyme and substrate at the same temperature and pH, but the substrate must have an excessive amount.

Chemical reactions can be studied by measuring the disappearance of the substrate, the rate of appearance by the product, or measuring the release or the absorbence of heat. For example, hydrogen peroxide (H2O2) is converted to water (H2O) and oxygen (O2) gas. Catalysis speeds up the reaction and sulfuric acid (H2SO4) stops the reaction by lowering the pH and denaturalizing the enzyme. Potassium permanganate (KmnO4) measure the presence of H2O2:
5 H2O2 + 2 KmnO4 +3 H2SO4 → K2SO4 + 2 MnSO4 + 8 H2O + 5 O2

After a certain amount of KmnO4 is added and the substance reaches a permanent brown or pink, no more KmnO4 should be added because it can mot be broken down.

Hypothesis:

Under perfect conditions, the rate of enzyme-catalysis should denature most of the hydrogen peroxide in a short amount of time.

Materials:

Exercise 2A

In Part 1, 10 mL of 1.5% of H2O2, a 50 mL beaker, and 1 mL of catalysis are needed. In Part 2, 5mL of catalysis, a water bath, and 10 mL of 1.5% H2O2 is needed. In Part 3, a 1 cm³ of liver, 50 mL beaker, and 10 mL of 1.5% H2O2. For all three parts, safety goggles, lab aprons, pencil, paper, erasers, and paper towels are needed.

Exercise 2B

To do this experiment, 10 mL of 1.5% H2O2, 1 mL of water, 10 mL of H2SO4, 50 mL beaker, 25 mL beaker, 5 mL syringe, and KmnO4 are needed. Safety goggles, lab aprons, pencil, paper, erasers, and paper towels are also needed.

Exercise 2C

To do this exercise, safety goggles, lab aprons, pencil, paper, erasers, paper towels, about 20 mL of 1.5% H2O2, 1 mL of H2O, 10 mL of H2SO4, 50 mL beaker, 25 mL beaker, 5 mL syringe, and KmnO4 are needed.

Exercise 2D

To do this experiment, about 60 mL of 1.5% H2O2, 6 mL of catalysis, 60 mL of H2SO4, 12 cups labeled 10, 30, 60, 120, 180, and 360 seconds, six cups labeled acid, a black marker, and a timer are needed. Safety goggles, lab aprons, pencil, paper, erasers, and paper towels will be needed, also.

Methods: * Remember to wear the goggles and apron. *

Exercise 2A

In Part 1, transfer 10 mL of 1.5% H2O2 a 50 mL glass beaker and add 1 mL of freshly made catalase to the solution. Remember to keep the catalase solution on ice at all times. Record the observations made. In Part 2, transfer 5 mL of the purified catalase extract to a test tube and place it in a boiling water bath for five minutes. Next, transfer 10 mL of 1.5% H2O2 into a 50 mL beaker and add 1 mL of the cooled, boiled catalase solution. Again record the results. In Part 3, cut 1 cm of liver, transfer it into a 50 mL glass beaker containing 10 mL of 1.5% H2O2, and mix it. Record the results.

Exercise 2B

To form a baseline for this experiment, put 10 ml of 1.5% H2O2 into a clean glass beaker. Add 1 mL of H2O and then add 10 mL of H2SO4 (1.0 M). Be careful when using acid. Mix this solution well. Remove a 5 mL sample and place it into another beaker. Assay for the amount of H2O2 as follows. Place the beaker containing the sample over white paper and use a 5 mL syringe to add one drop of KMnO4 at a time to the solution until it becomes a persistent pink or brown color. Gently swirl the solution after adding each drop. Record all results.

Exercise 2C

To determine the rate of spontaneous conversion of H2O2 to H2O and O2 in an uncatalyzed reaction, put about 20 mL of 1.5% H2O2 in a beaker. Store it uncovered at room temperature for approximately 24 hours. Put 10 ml of 1.5% H2O2 into a clean glass beaker (using the uncatalyzed H2O2 that set out). Add 1 mL of H2O and then add 10 mL of H2SO4 (1.0 M). Be careful when using acid. Mix this solution well. Remove a 5 mL sample and place it into another beaker. Assay for the amount of H2O2 as follows. Place the beaker containing the sample over white paper and use a 5 mL syringe to add one drop of KMnO4 at a time to the solution until it becomes a persistent pink or brown color. Gently swirl the solution after adding each drop. Record all results.

Exercise 2D

If a day or more has passed since Exercise B was performed, it is necessary to reestablish the baseline. Repeat the assay from Exercise B and record the results. Compare with other groups to check that results are similar. To determine the course of an enzymatic reaction, how much substrate is disappearing over time must be measured. The first thing to be done is to set up the cups labeled with times and acid. Add 10 mL of H2SO4 to each of the cups marked acid. Then put 10 mL of 1.5% H2O2 into the cup marked 10 sec. Add 1 mL of catalase extract to this cup. Swirl gently for 10 seconds (use the timer for accuracy). At 10 seconds, add the contents of one of the acid filled cups. Remove 5 mL and place in the second cup marked 10 sec. Assay the 5 mL sample by adding one drop of KMnO4 at a time until the solution turns a pink or brown. Repeat the above steps except allow the reactions to proceed for 30, 60, 120, 180, and 360 seconds, respectively. Use the times corresponding with the marked cups. Record all results and observations.

Results:

Table 1     Catalysis Activity

Table 2     Baseline Assay

Table 3     The Uncatalyzed Rate of H2O2 Decomposition

Table 4     New Baseline

Table 5     Catalyzed Rate of H2O2 Decomposition

Graph 1    Affect of Time on Enzyme-Catalyzed H2O2 (Remaining amount)

Exercise 2A

(a) What is the enzyme in this reaction?

The enzyme in the reaction is catalase.

(b) What is the substrate in this reaction?

The substrate in the reaction is hydrogen peroxide.

(c) What is the product in this reaction?

The products in the reaction are water and oxygen gas.

(d) How could you show that the gas evolved is O2?

The formula 2 H202 + catalase →2 H2O + O2 proves that water and oxygen gas can only be produced.

How does the reaction compare to the one using unboiled catalysis? Explain the reason for the difference.

The boiled catalysis was not as reactive as the regular catalysis, because boiling the catalysis denatures it.

What do you observe? What do you think would happen if the liver was boiled before being added to the H2O2?

The liver has a high amount of catalase in it causing it to be very reactive when put with hydrogen peroxide. If the liver was boiled first, the catalysis would have been denatured and would not have reacted as much as previously.

Exercise 2D

1) From the formula described earlier recall that rate = G y/G x. Determine the initial rate of the reaction and the rates between each of the time points. Record the rates in the table below.

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

The rate is the highest at initial to 10, because of the high concentration of catalysis.

When is the rate the lowest? For what reason is the rate low?

The rate is the lowest at 30 to 60 seconds, because the concentration of calase and the concentration of the product are beginning to balance each other out.

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

The sulfuric acid changes the pH of the catalase function and causes it to denature. Most enzymes work in a range of 6 to 8 and by adding the acid, the pH drops too low.

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

Lowering the temperature would slow the reaction. If the temperature is lowered a great deal (below 40ºC) it will be denatured.

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

To test the effect of temperature on enzymes: put 5 mL of catalase in the freezer until it is completely frozen, add 1 mL of catalase to 10 mL of 1.5% H2O2 (which is in a 50 mL beaker). Watch and record results.

Error of Analysis:

Errors in this experiment could have come from inaccurate measurements and timing. Also the catalase not being frozen when received most likely affected the data.

Discussion and Conclusion:

The purpose of this lab was to show the decomposition of hydrogen peroxide under different circumstances. Exercise 2A showed the affects of catalysis (added to and from living cells) in hydrogen peroxide. In Exercise 2B, the baseline was determined for the experiment (3.5). In Exercise 2C, the natural decomposition of hydrogen peroxide was viewed and found to be slower that when catalyzed. In Exercise 2D, a new baseline was made (3.6) and the decomposition of hydrogen peroxide with a catalase, over six minutes, was discovered to decompose more rapidly than an uncatalyzed reaction over 24 hours.

 

Lab 2    Enzyme Catalysis

 

Introduction:

Enzymes are proteins produced by living cells. They are biochemical catalysts meaning they lower the activation energy needed for a biochemical reaction to occur. Because of enzyme activity, cells can carry out complex chemical activities at relatively low temperatures. The substrate is the substance acted upon in an enzyme-catalyzed reaction, and it can bind reversibly to the active site of the enzyme. The active site is the portion of the enzyme that interacts with the substrate so that any substrate that blocks or changes the shape of the active sit effects the activity of the enzyme. The result of this temporary union is a reduction in the amount of energy required to activate the reaction of the substrate molecule so that products are formed. The following equation demonstrates this process: E + S ↔ ES ↔ E + P Enzymes follow the law of mass reaction. Therefore, the enzyme is not changed in the reaction and can be recycled to break down additional substrate molecules.

Several factors can affect the action of an enzyme: salt concentration, pH of the environment, temperature, activations and inhibitors. If salt concentration is close to zero, the changed amino acid side chains of the enzyme molecules will attract one another. The enzyme will then denature and form an inactive precipitate. Denaturation occurs when excess heat destroys the tertiary structure of proteins. This usually occurs at 40 to 50º Celsius. If salt concentration is high, the normal interaction of charged groups will be blocked. An intermediate salt concentration is normally the optimum for enzyme activity. The salt concentration of blood and cytoplasm are good examples of intermediate concentrations. The pH scale is a logarithmic scale that measures the acidity or H+ concentration in a solution and runs from 0 to 14, with 0 being highest in acidity and 14 lowest. Amino acid side chains contain groups such as –COOH that readily gain or lose H+ ions. As the pH is lowered an enzyme will tend to gain H+ ions, disrupting the enzyme’s shape. If the pH is raised, the enzyme will lose H+ ions and eventually lose its active shape. Reactions usually perform optimally in neutral environments. Chemical reactions generally speed up as the temperature is raised. More of the reacting molecules have enough kinetic energy to undergo the reaction as the temperature increases. However, if the temperature goes above the temperature optimum, the conformation of the enzyme molecules is disrupted. An activator is a coenzyme that increases the rate of the reaction and can regulate how fast the enzyme acts. It also makes the active site a better fit for the substrate. An inhibitor has the same power of activator regulation but decrease the reaction rate. An inhibitor also reduces the number of S-S bridges and reacts with the side chains near activation sites, blocking them.

The enzyme used in this lab is catalase. It has four polypeptide chains that are each composed of more than 500 amino acids. One catalase function is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes. Many oxidation reactions that occur in cells involve catalase. The following is the primary reaction catalyzed by catalase, the decomposition of hydrogen peroxide to form water and oxygen:

2 H2O2 → 2 H2O + O2 (gas) Without catalase this reaction occurs spontaneously but very slowly. Catalase speeds up the reaction notably.

The direction of an enzyme-catalyzed reaction is directly dependent on the concentration of enzyme, substrate, and product. For example, lots of substrate with a little product makes more product. Another example is lots of product with a little enzyme forms more substrate. Much can be learned about enzymes by studying the kinetics of enzyme-catalyzed reaction. It is possible to measure the amount of product formed, or the amount of substrate used, from the moment the reactants are brought together until the reaction has stopped.

Hypothesis:

Enzyme catalase, when working under optimum conditions, noticeably increases the rate of hydrogen peroxide decomposition.

Materials:

Exercise 2A

The materials needed for exercise 2A of the lab are: 30 mL of 1.5% (0.44 M) H2O2, a 50- mL glass beaker, 6 mL of freshly made catalase solution, a test tube, boiling water bath, 1 cm³ of liver, a knife for maceration, paper towels, safety goggles, lab apron, pencil, eraser, and paper to record results.

Exercise 2B

The materials needed for exercise 2B are: 10 mL of 1.5% H2O2, two clean glass beakers, 1 mL of H2O, 10 mL of H2SO4, a white sheet of paper, a 5 mL syringe, approximately 5 mL of KMnO4, paper, pencil, eraser, safety goggles, and lab aprons.

Exercise 2C

The materials needed for exercise 2C of the lab are: 20 mL of 1.5% H2O2, two glass beakers, 1 mL of H2O, 10 mL of H2SO4, a white sheet of paper, a 5 mL syringe, approximately 5 mL of KMnO4, paper, pencil, eraser, safety goggles, and lab aprons.

Exercise 2D

For this part of the experiment, the materials needed are 12 cups labeled 10, 30, 60, 120, 180, and 360 on two each, six cups labeled acid, 60 mL of 1.5% H2O2, a clean 50-mL beaker, 6 mL of catalase extract, two 5-mL syringes, KMnO4, a timer, paper, pencil, black marker, eraser, safety goggles, and lab aprons.

Methods:

Exercise 2A

Transfer 10 mL of 1.5% H2O2 into a 50-mL glass beaker and add 1 mL of freshly made catalase solution. Remember to keep the catalase solution on ice at all times. Record the results. Then transfer 5 mL of purified catalase extract to a test tube and place it in a boiling water bath for five minutes. Transfer 10 mL of 1.5% H2O2 into a 50-mL beaker and add 1 mL of the cooled, boiled catalase solution. Again record the results. To demonstrate the presence of catalase in living tissue, cut 1 cm of liver, macerate it, and transfer it into a 50-mL glass beaker containing 10 mL of 1.5% H2O2. Record these results.

Exercise 2B

Put 10 ml of 1.5% H2O2 into a clean glass beaker. Add 1 mL of H2O. Add 10 mL of H2SO4 (1.0 M) using extreme caution. Mix this solution well. Remove a 5 mL sample and place it into another beaker. Assay for the amount of H2O2 as follows. Place the beaker containing the sample over white paper. Use a 5-mL syringe to add KMnO4 a drop at a time to the solution until a persistent pink or brown color is obtained. Remember to gently swirl the solution after adding each drop. Record all results. Check with another group before proceeding to see that results are similar.

Exercise 2C

To determine the rate of spontaneous conversion of H2O2 to H2O and O2 in an uncatalyzed reaction, put about 20 mL of 1.5% H2O2 in a beaker. Store it uncovered at room temperature for approximately 24 hours. Repeat the steps from Exercise 2B, using the uncatalyzed H2O2, to determine the proportional amount H2O2 of remaining after 24 hours. Record the results.

Exercise 2D

If a day or more has passed since Exercise B was performed, it is necessary to reestablish the baseline. Repeat the assay and record the results. Compare with other groups to check that results are similar. To determine the course of an enzymatic reaction, how much substrate is disappearing over time must be measured. First, set up the cups with the times and the word acid up. Add 10 mL of H2SO4 to each of the cups marked acid. Then put 10 mL of 1.5% H2O2 into the cup marked 10 sec. Add 1 mL of catalase extract to this cup. Swirl gently for 10 seconds. (Calculate time using the timer for accuracy.) At 10 seconds, add the contents of one of the acid filled cups. Remove 5 mL and place in the second cup marked 10 sec. Assay the 5-mL sample by adding KMnO4 a drop at a time until the solution obtains a pink or brown color. Repeat the above steps except allow the reactions to proceed for 30, 60, 120, 180, and 360 seconds, respectively. Use the times’ corresponding, marked cups. Record all results and observations.

Results:

Table 1: Test of Catalysis Activity

Table 2: Establishing a Baseline #1

Table 3: Uncatalyzed H2O2 Decomposition

Table 4: Establishing a Baseline #2

Table 5: Time-Course Determination

Effect of Time on the Amount of H2O2 Remaining after an Enzyme Catalyzed Reaction

Exercise 2A:

1.a. What is the enzyme in this reaction? The enzyme in this reaction is the catalase solution.

1.b. What is the substrate in this reaction? The substrate is hydrogen peroxide.

1.c. What are the products in this reaction? The products are water and oxygen gas.

1.d. How could you show that the gas evolved is oxygen? Referring to the equation 2H2O2 + Catalase solution→H2O + O2, the only gas released is oxygen.

2. How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference. With the boiled catalase, there was no sign of bubbling because the catalase was denatured by the heat and caused no reaction.

3.a. What do you observe? I observe quite a bit of gas being released from the solution.

3.b. What do you think would happen if the liver were boiled before being added to the hydrogen peroxide? I think that no signs of a reaction occurring would be shown. The catalase that occurs naturally within the liver would have been denatured.

4. From the formula described earlier recall that rate = G y/G x . Determine the initial rate of the reaction and the rates between each of the time points. Record the rates in the table below.

5. When is the rate the highest? Explain why. The rate is the highest in the first ten seconds because the rate decreases as the concentration of the catalase decreases over time.

6. When is the rate the lowest? For what reason is the rate low? The rate is lowest during the last time period of 360 seconds because the most time has passed. The catalase concentration has been reduced and the product amount has increased, blocking the enzymes from reacting with the hydrogen peroxide.

7. Explain the inhibiting effect of sulfuric acid on the function of the catalysis. Relate this to enzyme structure and chemistry. The sulfuric acid’s high concentration of H+ ions gives the acid a low pH. Because enzymes can only function in the pH range of six to eight, the addition of an acidic solution denatures the enzyme, stopping the reaction.

8. Predict the effect of lowering the temperature would have on the rate of the enzyme activity. Explain your prediction. Enzymes generally only work at the between the temperatures of forty and fifty degrees Celsius. Lowering the temperature would slow the reaction until the enzyme is denatured and no longer able to react.

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

Part One (the effects of a strong acid on enzyme activity): Add 10 mL of 1.5-% hydrogen peroxide to a 50-mL beaker, and add 1 mL of catalase solution. Mix well and then add 1 mL of (0.5 M) HCl to the beaker. Observe the reaction and record the results.

Part Two (the effects of a neutral solution on enzyme activity): Add 10 mL of 1.5-% hydrogen peroxide to a 50-mL beaker, and add 1 mL of catalase solution. Mix well and then add 1 mL of pure water with a pH of 7.0. Observe the reaction and record the results.

Part Three (the effects of a strong base on enzyme activity): Add 10 mL of 1.5-% hydrogen peroxide to a 50-mL beaker, and add 1 mL of catalase solution. Mix well and then add 1 mL of (0.5 M) NaOH to the beaker. Observe the reaction and record the results.

Error Analysis:

Several errors could have occurred throughout the experiment. Miscalculations involving numbers and amounts of solutions would have a severe effect upon the results. Mathematical errors may also have of occurred. When the catalase arrived, it had melted. Because it is to remain on ice at all times, this may have caused errors. The age of the hydrogen peroxide effected results. For example, when calculating the percent of hydrogen peroxide spontaneously decomposed after 24 hours, new hydrogen peroxide yielded a much higher percentage than the aged hydrogen peroxide. Errors occur in every experiment and that is why is it is necessary to repeat an experiment several times for the most accurate results.

Discussion and Conclusion:

Catalase, or enzymes, drastically increases the rate of hydrogen peroxide decomposition. This lab shows how catalase added to hydrogen peroxide leads to the release of oxygen, boiled catalase is denatured, and the presence of catalase in living things can lead to the breaking down of hydrogen peroxide in the body. In the lab it was shown that the natural decomposition hydrogen peroxide is slower than decomposition taking place with the addition of enzymes. If hydrogen peroxide was required to decompose naturally, life could not survive. The addition of catalase increases this decomposition rate allowing life to continue.

Lab. 2 – Enzyme Catalysis

Introduction:

This lab will observe the conversion of hydrogen peroxide to water and oxygen gas by the enzyme catalysis. The amount of oxygen generated will be measured and used to calculate the rate of the enzyme-catalized reaction. Enzymes are proteins produced by living cells. Enzymes act as biochemical catalysts during a reaction, meaning they lower the activation energy needed for that reaction to occur. Through enzyme activity, cells gain the ability to carry out complex chemical activities at relatively low temperatures. The substance in an enzyme-catalyzed reaction that is to be acted upon is the substrate, which binds reversibly to the active site of the enzyme. The active site is the portion of the enzyme that interacts with the substrate. One result of this temporary union between the substrate and the active site is a reduction in the activation energy required to start the reaction of the substrate molecule so that products are formed. In a mathematical equation of the substrate (S) binding with the activation site (E) and forming products (P) is:

E + S —> ES –> E + P

Several ways enzyme action may be affected include:

1) Salt Concentration —  For example, if salt concentration is close to zero, the charged amino acid side chains of the enzyme molecules will attract each other. The enzyme will then denature and form an inactive precipitate. If salt concentration is extremely high, the normal interaction of charged groups will be blocked, new interactions will occur, and again the enzyme will precipitate.  An intermediate salt concentration such as that of human blood (0.9%) is the optimum for many enzymes.

2) pH of the environment — . The pH of a solution is a logarithmic scale that measures the acidity or H+ concentration in a solution.  The scale begins at 0, being the highest in acidity, and ends at 14, containing the least amount of acidity. As the pH is lowered an enzyme will tend to gain H+ ions, disrupting the enzyme’s shape. In turn, if the pH is raised, the enzyme will lose H+ ions and eventually lose its active shape.

3) Temperature — Usually, chemical reactions speed up as the temperature is raised. When the temperature is increased, more of the reacting molecules have enough kinetic energy to undergo the reaction. However, if the temperature goes past a temperature optimum, the conformation of the enzyme molecules is disrupted.

4) Activations and Inhibitors — Many molecules other than the substrate may interact with an enzyme. If such a molecule speeds up the reaction it is an activator, but if it slows the reaction down it is an inhibitor.

The enzyme used in this lab is catalase, which has four polypeptide chains that are composed of more than 500 amino acids each. One function of this enzyme is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes. Catalase is also involved in some of the many oxidation reactions that occur in the cells of all living things. The primary reaction catalyzed by catalase is the decomposition of hydrogen peroxide to form water and oxygen.

2H2O2  ——->  2 H2O  +  O2  (gas)

Without the help of catalase, this reaction occurs spontaneously, but very slowly. Catalase helps to speed up the reaction considerably. In this lab, a rate for this reaction will be determined.

Hypothesis:

The enzyme catalase, under optimum salt conditions, temperature, and pH level will speed up the reaction as it denatures the hydrogen peroxide at a higher rate than normal.

Materials:

Exercise 2A

For the first part of the lab, 10 mL of 1.5% H2O2, a 50-mL glass beaker, and 1 mL of fresh catalase are needed. At the second stage a test tube, a hot water bath, 5 mL of catalase, 10 mL of 1.5% H2O2 are needed. Finally, in the third part, a potato, and10 mL of 1.5% H2O2 are needed.

Exercise 2B

For this experiment, 10 mL of 1.5% H2O2, 1 mL of water, 10 mL of sulfuric acid, two 25 mL beakers, 5-10 mL syringe, potassium permanganate, lab aprons and trays are needed.

Exercise 2C

In this section of the experiment, 20 mL of 1.5% H2O2, two glass beakers, 1 mL of H2O, 10 mL of sulfuric acid, a 5 mL syringe, 5-10 mL of potassium permanganate, and lab aprons and trays are used.

Exercise 2D

In the final part of the lab, 6 plastic cups labeled 10, 30, 60, 120, 180, 360, 6 plastic cups labeled acid, 60 mL of 1.5% H2O2, a 50-mL beaker, 6 mL of catalase extract, two 5-mL syringes, potassium permanganate, a timer (clock), lab aprons and trays are needed.

Methods:

Exercise 2A

Transfer 10 mL of 1.5% H2O2 into a 50-mL glass beaker and add 1 mL of freshly made catalase solution. The fresh catalase should be kept on ice until ready to be used. Observe the reaction. Then transfer 5 mL of purified catalase extract to a test tube and place it in a hot water bath for five minutes. Transfer 10 mL of 1.5% H2O2 into a 50-mL beaker and add 1 mL of the boiled catalase solution, after it has cooled. Observe the changes in the reaction. To demonstrate the presence of catalase in living tissue, cut 1 cubic cm of potato, macerate it, and transfer it into a 50-mL glass beaker containing 10 mL of 1.5% H2O2. Observe the results.

Exercise 2B

Put 10 ml of 1.5% H2O2 into a clean glass beaker. Add 1 mL of H2O. Add 10 mL of sulfuric acid (1.0 M). USE EXTREME CARE IN HANDLING ACIDS. Mix the solution well. Remove a 5 mL sample. Place this 5 mL sample in another beaker, and assay for the amount of H2O2 as follows: Place the beaker containing the sample over white paper. Use a burette or 5 mL pipette to add potassium permanganate a drop at a time to the solution until a persistent pink or brown color is obtained. Remember to gently swirl the solution after adding each drop.

Exercise 2C

To determine the rate of spontaneous conversion of H2O2 to H2O and O2in an uncatalyzed reaction, put about 20 mL of 1.5% H2O2 in a beaker. Store it uncovered at room temperature for approximately 24 hours. Put 10 mL of 1.5% H2O2 into a clean glass beaker (using the uncatalyzed H2O2 that set out). Add 1 mL of H2O2 and then add 10 mL of sulfuric acid (1.0 M). Be careful when using acid. Mix this solution well. Remove a 5 mL sample and place it into another beaker. Assay for the amount of H2O2 as follows: Use a 5 mL syringe to add one drop of potassium permanganate at a time to the solution until it becomes a persistent pink or brown color. Gently swirl the solution after adding each drop. Record all results.

Exercise 2D

If a day or more has passed since Exercise B was performed, a baseline must be reestablish. Repeat the assay from Exercise B and record the results. Compare with other groups to check that results are similar. To determine the course of an enzymatic reaction, how much substrate is disappearing over time must be measured. The first thing to be done is to set up the six cups labeled with times, and the other six, one directly in front of each cup with a time on it. Then put 10 mL of 1.5% H2O2 into the cup marked 10 sec. Add 1 mL of catalase extract to this cup. Swirl gently for 10 seconds using a timer or clock for help. At 10 seconds, add 10 mL of sulfuric acid. Remove 5 mL and place in the cup directly in front of the cup marked 10 sec. Assay the 5 mL sample by adding one drop of potassium permanganate at a time until the solution turns a pink or brown. Repeat the previous steps, with clean cups using the times 30, 60, 120, 180, and 360. Record all results and observations.

Results:

Table 1

Enzyme Activity

 Table 2

Establishing a Baseline

Table 3

Rate of Hydrogen Peroxide Spontaneous Decomposition

Table 4

Reestablishing a Baseline

Table 5

Rate of Hydrogen Peroxide Decomposition by Catalase

Graph 1

Exercise 2A

1. a) What is the enzyme in this reaction? Catalase is the enzyme in the reaction.

    b) What is the substrate in this reaction? The substrate is hydrogen peroxide.

 c) What is the product in this reaction? The products are oxygen (gas) and water.

d) How could you show that the gas evolved is oxygen? Using the example of holding a                                                                                                                                                                                                 burning wooden splint over the reaction, the splint glows bright red, therefore showing that oxygen is being let out of the solution.

2. How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference.  When the boiled catalase was used, there was no bubbling in the solution, which proved that there was no reaction occurring because the extreme heat had denatured the catalase.

3. What do you observe? What do you think would happen if the potato or liver was boiled before being added to the hydrogen peroxide?  The catalase shows a lot of reaction with the potato, causing many bubbles to form in the solution. Also, if the potato were boiled there wouldn’t be any bubbles, because the heat would denature the potato.

Analysis of Results

1. Determine the initial rate of the reaction and the rates between each of the time points. Record the rates in the table below.

2. When is the rate the highest? Explain why.  The rate is the highest at the beginning of the reaction, because the hydrogen peroxide had been exposed to the air for the least amount of time.

3. When is the rate the lowest? For what reasons is the rate low?  At the longer times the rate was the lowest because the peroxide had been exposed to the air longer.

4. Explain the inhibiting effect of sulfuric acid on the function of catalase. Relate this to enzyme structure and chemistry.  The sulfuric acid lowered the pH level of the solution, which caused the catalase to denature by gaining hydrogen ions and it stopped the reaction immediately.

5. Predict the effect lowering the temperature would have on the rate of enzyme activity. Explain you prediction.  Enzymes work best at optimum temperature, therefore increasing, or in this case, decreasing the temperature would extremely change the rate of the reaction. Lowering the temperature would cause the reaction to slow down.

6. Design a controlled experiment to test the effect of varying pH, temperature, or enzyme concentration.  Since the results of room temperature and heated have already been recorded, using catalase that was completely frozen would test the other end of the spectrum as far as temperature goes.

Error Analysis:

Any errors occurring in this experiment could have been caused by misreading of a syringe, miscalculating the data on the tables, or when the 1.5% H2O2 was mixed that was used in almost all parts of the experiment. Also, in Exercise 2D, if the getting the timing just right with all parts of the lab were a source of error in the experiment.

Discussion and Conclusion:

The main purpose of this lab was to show how enzymes can be affected in reactions with other substances by factors such as pH, temperature, and exposure to the surrounding environment. This lab proved that an extreme increase in temperature (boiling) can cause absolutely no reaction by using the boiled enzyme catalase. Also, by using a potato, it shows that catalase is speeding up the decomposition of hydrogen peroxide in living things, helping all living things survive another day.

AP Biology: CHAPTER 5A

MACROMOLECULES

1. Define the following:

a. monomer _______________________________________________________________

___________________________________________________________________________

b. polymer _________________________________________________________________

___________________________________________________________________________

c. condensation reaction _____________________________________________________

___________________________________________________________________________

d. hydrolysis _______________________________________________________________

___________________________________________________________________________

2. Which foods do you think will enter the blood the quickest? Why?

___________________________________________________________________________

___________________________________________________________________________

3. What are the general roles of carbohydrates? ______________________________________

___________________________________________________________________________

___________________________________________________________________________

4. List some monosaccharides with their molecular formulas.

___________________________________________________________________________

___________________________________________________________________________

5. Double sugars are called ______________________________________________________

6. List the monosaccharides that form each:

a. maltose _________________________________________________________________

b. sucrose _________________________________________________________________

c. lactose __________________________________________________________________

6. Polymers of sugars form _______________________________________________________

7. Which forms of polysaccharide is best for each function:

a. Strength of structure _______________________________________________________

b. Storage and sugar release __________________________________________________

c. What theme is this addressing? ______________________________________________

8. How does the alpha differ from the beta form of glucose and why is it significant to animals?

___________________________________________________________________________

___________________________________________________________________________

9. How do the role and structure of the following polysaccharides compare?

a. starch___________________________________________________________________

b. glycogen ________________________________________________________________

c. cellulose_________________________________________________________________

10. Ninety percent of Asians, 75% of African-Americans, and a much smaller percent of

northern Europeans are lactose intolerant. Why do you suppose we see this pattern?

___________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

AP Biology: CHAPTER 5B

MACROMOLECULES

1. What is the characteristic common to lipids? _______________________________________

___________________________________________________________________________

2. Lipids are synthesized by the chemical reaction ____________________________________

and broken down by the reaction ________________________________________________

3. What makes fats hydrophobic? _________________________________________________

___________________________________________________________________________

___________________________________________________________________________

4. State at least two differences between saturated and unsaturated fats.

a. _________________________________________________________________________

___________________________________________________________________________

b. _________________________________________________________________________

___________________________________________________________________________

5. How do phospholipids interact in an aqueous solution?

___________________________________________________________________________

___________________________________________________________________________

6. Make a diagram of phospholipid interactions that form membranes.

7. Sketch the common building block of steroids.

8. List several functions of proteins.

___________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

9. What are the three properties used to classify amino acids?

___________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

10. Sketch two amino acids side-by-side, on one of them label the functional groups, then show how the two can be joined together.

11. What determines the primary structure of a protein? _________________________________

___________________________________________________________________________

12. Describe the four levels of protein structure:

a. Primary _________________________________________________________________

___________________________________________________________________________

b. Secondary ______________________________________________________________

___________________________________________________________________________

c. Tertiary _________________________________________________________________

___________________________________________________________________________

d. Quaternary ______________________________________________________________

___________________________________________________________________________

13. What happens to a protein during denaturation? ____________________________________

___________________________________________________________________________

14. What are the building blocks of nucleic acids? ______________________________________

15. Briefly describe two functions of DNA in the cell.

a. ________________________________________________________________________

___________________________________________________________________________

b. ________________________________________________________________________

___________________________________________________________________________