Fall 1997


Laboratories 3 & 4 - A Quantitative Enzyme Study; CATALASE

I. INTRODUCTION

Enzymes are biological catalysts that carry out the thousands of chemical reactions that occur in living cells. They are generally large proteins made up of several hundred amino acids, and often contain a nonproteinaceous group, called the prosthetic group, that is important in the actual catalysis.

In an enzyme-catalyzed reaction, the substance to be acted upon, or substrate, binds to the active site of the enzyme. The enzyme and substrate are held together in an enzyme-substrate complex by hydrophobic bonds, hydrogen bonds and ionic bonds.

The enzyme then converts the substrate to the reaction products in a process that often requires several chemical steps, and may involve covalent bonds. Finally, the products are released and the enzyme is ready to form another enzyme-substrate complex. As is true of any catalyst, the enzyme is not used up as it carries out the reaction, but is recycled again and again. One enzyme molecule can carry out thousands of reaction cycles every minute.

Each enzyme is specific for a certain reaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure. The business portion of the enzyme molecule, the active site, also has a specific shape so that only one or a few of the thousands of compounds present in the cell can interact with it. Any substance that blocks or changes the shape of the active site will interfere with the activity and efficiency of the enzyme. If these changes are large enough, the enzyme can no longer act at all, and is said to be denatured. There are several factors that are especially important in determining the enzyme's shape, and these are closely regulated both in the living organism and in laboratory experiments to give the optimum enzyme activity:

  1. Salt concentration. If the salt concentration is very low or zero, the charged amino acid side chains of the enzyme will stick together. The enzyme will denature and form an inactive precipitate. If, on the other hand, the salt concentration is very high, normal interaction of charged groups will be blocked, new interactions occur, and again the enzyme will precipitate. An intermediate salt concentration such as that of blood (0.9% w/v) or cytoplasm is optimum for most enzymes.

  2. pH. pH is a logarithmic scale that measures the H+ concentration in a solution. The scale runs from 0 to 14 with 0 being the highest in acidity and 14 the lowest. Neutral solutions have a pH of 7. Acid solutions have a pH less than 7; basic solutions have a pH greater than 7. Enzyme amino acid side chains contain groups such as -COOH and -NH2 that readily gain or lose H+ ions. As the pH is lowered, an enzyme will tend to gain H+ ions, and eventually enough side chains will be affected so that the enzyme's shape is disrupted. Likewise, as the pH is raised, the enzyme will lose H+ ions and eventually lose its active shape. Many enzymes have an optimum in the neutral pH range and are denatured at either extremely high or low pH. Some enzymes, such as those which act in the human stomach where the pH is very low, will have an appropriately low pH optimum. A buffer is a compound that will gain or lose H+ ions so that the pH of a solution changes very little.

  3. Temperature. All chemical reactions speed up as temperature is raised. As the temperature increases, more molecules have enough kinetic energy to undergo the reaction. Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to proceed faster with increasing temperature. However, if the temperature of an enzyme-catalyzed reaction is raised still further, an optimum is reached: above this point, the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme molecules starts to be disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of denaturing more and more enzyme molecules. Many proteins are denatured by temperatures around 40-50deg.C, but some are still active at 70-80deg.C, and a few even withstand being boiled.

  4. Small molecules. Many molecules other than the substrate may interact with an enzyme. If such a molecule increases the rate of the reaction it is an activator, and if it decreases the reaction rate it is an inhibitor. The cell can use these molecules to regulate how fast an enzyme acts. Any substance that tends to unfold the enzyme, such as an organic solvent or detergent, will act as an inhibitor. Some inhibitors act by reducing the -S-S- bridges that stabilize the enzyme's structure. Many inhibitors act by reacting with side chains in or near the active site to change or block it. Others may damage or remove the prosthetic group. Many well known poisons, such as potassium cyanide and curare, are enzyme inhibitors which interfere with the active site of a critical enzyme.

In this exercise you will study the enzyme catalase, which accelerates the breakdown of hydrogen peroxide (a common end product of oxidative metabolism) into water and oxygen, according to the summary reaction:

2H2O2 + catalase ----> 2H2O + O2 + catalase

This catalase-mediated reaction is extremely important in the cell because it prevents the accumulation of hydrogen peroxide, a strong oxidizing agent which tends to disrupt the delicate balance of cell chemistry.

Catalase is found in animal and plant tissues, and is especially abundant in plant storage organs such as potato tubers, corms and in the fleshy parts of fruits. You will isolate catalase from potato tubers and measure its rate of activity under different conditions. A glass fiber filter will be immersed in the enzyme solution, then placed in the hydrogen peroxide substrate. The oxygen produced in the subsequent reaction will become trapped in the disc, making it buoyant. The time measured from the moment the disc touches the substrate to the time it reaches the surface of the solution is a measure of the rate of the enzyme activity.

II. PROCEDURE

A. Extraction of catalase

  1. Peel a fresh potato tuber and cut the tissue into small cubes. Weigh out 100 gm of tissue.

  2. Place the tissue, 100 ml of cold distilled water and a small amount of crushed ice in a prechilled blender.

  3. Homogenize for 30 seconds at high speed.

    FROM THIS POINT ON, THE ENZYME PREPARATION MUST BE CARRIED OUT IN AN ICE BATH

  4. Filter the potato extract, then pour the filtrate into a 250 ml graduated cylinder. Add cold distilled water to bring up the final volume to 200 ml, mix well and split into (2) 100 ml portions. This extract will be arbitrarily labeled 100 units of enzyme per ml (100 units/ml) and will be used in parts B & C. Repeat the extraction procedure next week for parts D, E, & F.

B. Effect of catalase concentration

Before considering the factors which affect enzyme reactions, it is important to demonstrate that the enzyme assay shows that the enzyme actually follows accepted chemical principles. One way to demonstrate this is by determining the effect of enzyme concentration on the rate of activity, while using a substrate concentration which is in excess.

Label five 50 ml beakers as follows: 100, 50, 25, 10 and 0 units/ml. Prepare 40 ml of enzyme for each of the above concentrations in the following manner:

ml orig. enzyme+ml cold distilled H2O=units/ml

40*0100
202050
103025
43610
0400

*Save this undiluted enzyme for part C.

Keep your catalase preparations in the ice bath. Label an identical set of beakers for the substrate. Into each of these beakers, measure out 40 ml of a 1% hydrogen peroxide solution/

Using forceps, immerse 1/4 of a 2.1 cm glass fiber filter disc in the catalase solution you have prepared. Allow the disc to absorb the enzyme solution for 5 seconds, remove and drain for 10 seconds on a paper towel. Drop the disc into the first substrate solution. The disc will sink rapidly into the solution. The oxygen produced from the breakdown of the hydrogen peroxide by catalase becomes trapped in the fibers of the disc causing the disc to float to the surface of the solution. The time (t) in seconds, from the second the disc touches the solution to the time it again reaches the surface is determined to be the rate (R) of enzyme activity where (R) = 1/(t sec). Carry out the procedure twice for each enzyme concentration and average the results.

C. Effect of substrate concentration

To determine the effect of substrate concentration on enzyme activity obtain eight 50 ml beakers and label them as follows:

0
0.1%H2O2
0.2%H2O2
0.5%H2O2
0.8%H2O2
1%H2O2
5%H2O2
10%H2O2

Add 40 ml of the proper (as outlined above) H2O2 solution to each beaker. Make sure that the substrate solutions reach room temperature before beginning your assay. Using the filter discs procedure described above, and the 100 units/ml enzyme solution from part B, determine the rate of the reaction at the various substrate concentrations. Record your results in the table provided. Carry out the procedure twice for each substrate concentration and average the results.

D. Effect of enzyme inhibition

Hydroxylamine attaches to iron atoms in the catalase molecule and thereby interferes with the formation of the enzyme-substrate complex. Obtain 5 ml of enzyme extract (100 units/ml). Label 4 Eppendorf tubes as follows: 1%, 0.1%, 0.01% and 0% (control). These percentages refer to the hydroxylamine solutions available for you to test. Into each of these 4 tubes, place 1 ml of the enzyme using a 1 ml pipette and Pi-pump. Add 2 drops of the appropriate hydroxylamine solution and use diH2O for the control. Gently flick each tube with your finger to mix and let the tubes stand for 1 minute. Set up corresponding plastic beakers with 40 ml of H2O2 and test as before using filter paper dipped in enzyme, blotted on paper towel and then dropped in H2O2. Test each enzyme solution with both 3% and 5% H2O2. Carry out the procedure twice for each solution and average the results. Is hydroxylamine a competitive inhibitor of the catalase? Why or why not?

E. Effect of temperature

Using 40 ml of a 1% hydrogen peroxide solution as the substrate, and 5 ml aliquots of the 100 units/ml enzyme solution, measure the enzyme activity in the usual manner. Run the reactions in the water baths at different temperatures, such as 4deg., 15deg., room temperature (which is about 22deg.), 30deg., and 37deg.. The catalase and substrate should be brought to the testing temperature before they are used. Record the exact temperature and your data in an appropriate table. Also test the activity of enzyme that has been boiled. [Do not boil the H2O2]. Carry out the procedure twice for each temperature and average the results.

F. Effect of pH

Obtain 5 50-ml beakers, and label them as follows:

Control, pH 4, pH 6, pH 8, pH 10

Into each beaker, pour 10 ml of 100 units/ml enzyme solution and 30 ml of buffer solution at the appropriate pH. (Use 30 ml dH2O for the control).
Using 40 ml of a 1% hydrogen peroxide solution as the substrate, measure the enzyme activity in the usual manner. Carry out the procedure twice for each pH and average the results.

III. ANALYSIS OF RESULTS

Prepare a written laboratory report following the "Scientific Writing" handout provided in lab. Use the following questions as a guide in preparing your Results and Discussion sections. Make sure to provide all information in the Discussion in paragraph, prose format - not just a series of answers to the questions. The lab report is due the week of Sept. 30 - October 3 during your regular lab meeting.

  1. Record your results from the effect of catalase concentration and plot them on a graph [enzyme activity (R) is the ordinate and catalase concentration is the abscissa], labeling it carefully. How does enzyme activity vary with enzyme concentration? Explain.

  2. Plot your results from the effect of substrate concentration on a graph. a) How is the rate of enzyme activity affected by increasing the concentration of the substrate? b) What do you think would happen if you increased the substrate concentration to 20% H2O2. c) Does changing the substrate concentration exhibit the same effect as changing the enzyme concentration?

  3. Explain the results from the effect of enzyme inhibition.

  4. From the data on the effect of temperature analysis, what can you conclude about how temperature affects enzyme activity? Plot the results on the graph. How would you explain the results?

  5. How does pH affect enzyme activity? Plot your results on the graph. Would you expect similar results with salivary amylase (an enzyme found in the mouth)? Pepsin (an enzyme found in the stomach)?