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Enzyme Lab
Background information
Potatoes and other living tissues contain the enzyme catalase. This enzyme breaks down hydrogen
peroxide, which is a harmful by-product of the process of cellular respiration. If we use potato
and other tissues containing this enzyme, we can measure the relative influence of several
different factors on the activity of enzymes in living tissue.
In order to obtain energy and building blocks from food, the digestive system breaks down
proteins, fats and carbohydrates into amino acids, fatty acids, and monosaccarides. In this
process, specific enzymes catalyze hydrolysis reactions in which food polymers are broken up into
monomers.
Objectives met: * Measure the effects of changes in temperature, pH, and enzyme
concentration on reaction rates of an enzyme catalyzed reaction in a controlled experiment.
* Explain how environmental factors affect the rate of enzyme-catalyzed reactions.
INTRODUCTION: What would happen to your cells if they made a poisonous chemical as a
byproduct? You might think that they would die. In fact, your cells are always making poisonous
chemicals. They do not die because your cells use enzymes to break down these poisonous
chemicals into harmless substances. Enzymes are proteins that speed up the rate of reactions
that would otherwise happen more slowly. The enzyme is not altered by the reaction. You have
hundreds of different enzymes in each of your cells.
Each of these enzymes is responsible for one particular reaction that occurs in the cell. In this
lab, you will study an enzyme that is found in the cells of many living tissues. The name of the
enzyme is catalase (KAT-uh-LAYSS); it speeds up a reaction which breaks down hydrogen
peroxide, a toxic chemical, into 2 harmless substances--water and oxygen.
The reaction is as follows: 2H2O2 ----> 2H2O + O2
This reaction is important to cells because hydrogen peroxide (H2O2) is produced as a byproduct
of many normal cellular reactions. If the cells did not break down the hydrogen peroxide, they
would be poisoned and die. In this lab, you will study the catalase found in potato.
The catalase enzyme is a tetramer of four polypeptide chains, each over 500 amino acids long. It
contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen
peroxide. There is an optimum pH for human catalase that is a fairly broad. The pH optimum for
other catalases varies depending on the species. The optimum temperature also varies by species.
MATERIALS:
1molar HCl solution (in dropper bottle)
1molar NaOH solution (in dropper bottle)
6 Test tubes and Test tube holder
10-ml Graduated cylinder
40 ml 3% Hydrogen peroxide solution
Straight-edged razor blade
Scissors and Forceps (tweezers)
Thermometer
Stirring rod
pH paper
Fresh potato
PART A – Observe Normal Catalase Reaction at Room Temperature
1. Place 2 ml of the 3% hydrogen peroxide solution into a clean test tube.
Using a knife cut a small piece of potato and add it to the test tube. Push it into the
hydrogen peroxide with a stirring rod. Observe the bubbles.
What gas is being released? Throughout this investigation you will estimate the rate of the
reaction (how rapidly the solution bubbles) on a scale of 0-5 (0=no reaction, 1=slow to 5= very
fast). Assume that the reaction in step 2 proceeded at a rate of "4".
A reaction that absorbs heat is endothermic; a reaction that gives off heat is exothermic. Now,
feel the temperature of the test tube with your hand. Has it gotten warmer or colder - Is the
reaction endothermic or exothermic? What is happening in your test tube?
Is Catalase Reusable?
2. Pour off the liquid into a second test tube. Assuming the first reaction is complete.
Consider: What is this liquid composed of? What do you think would happen if you added
more potato to this liquid?
Test this by adding more potato into the used catalase and record the reaction rate.
Explain your results (what is the liquid composed of and what happened?)
3. Add another 2 ml of hydrogen peroxide to the potato remaining in the first test tube.
Is catalase reusable?
PART B - What is the Effect of Temperature on Catalase Activity?
1. Put a piece of potato into the bottom of a clean test tube and cover it with a small amount of
distilled water. Place this test tube in a boiling water bath for 5 minutes.
What will boiling do to an enzyme?
2. Remove the test tube from the hot water bath, allow it to air cool, then pour out the water.
Add 2 ml of hydrogen peroxide. CAUTION: Use a test-tube holder when handling the hot test
tubes. Record the reaction rate (0-5) in DATA TABLE 1.
3. Put equal quantities of potato into 2 clean test tubes and 1 ml H2O2into 2 other test tubes. Put
one test tube of potato and one of H2O2 into each of the following water baths: Ice bath and
Warm water bath (not boiling). Record the temperature of the water baths in DATA TABLE 1.
4. After 3 minutes, pour each tube of H2O2 into the corresponding tube of potato and observe
the reaction. Record the reaction rates (0-5) in DATA TABLE 1.
You recorded the reaction rate for room temperature earlier. What is the "optimum"
temperature for catalase? (This is the temperature at which the reaction proceeds fastest.)
Why did the reaction proceed slowly at 0 deg.C? Why did the reaction not proceed at all at 100
deg.C?
PART C - What is the Effect of pH on Catalase Activity
CAUTION: Do not let acids or bases contact your skin or clothing. Swirl each test tube after
adding each drop and measure the pH of each solution with pH paper. To do this, remove a drop or
two of solution from a test tube using a clean glass stirring rod. Rinse your stirring rod and wipe
dry before you dip it into each test tube. Place the drop on pH paper. Record the pH of each
solution in DATA TABLE 1.
1. Add 2 ml hydrogen peroxide to each of 3 clean test tubes. Treat each tube as follows:
Tube 1--add a drop of HCl (acid) at a time until pH 3 or lower.
Tube 2--add a drop of NaOH (base) at a time until pH 10 or higher.
Tube 3—test pH
2. Once each tube has reached the desired pH place a potato in the tube and observe the
reaction. Record the reaction rates (0-5) in DATA TABLE 1.
Does there appear to be a pH "optimum" - at what pH? What is the effect of low or high pH on
enzyme activity?
Name:______________________________________ Date:___________________ Period:________
DATA TABLE 1
Part
Material Tested
PART A
Normal Potato
Reaction
Rate (0-5)
Observations
Potato added to Used
Peroxide
Reused Catalase
Part B
Boiled Potato at _______(oC)
Ice Bath Potato at ______(oC)
Warm Potato at _______(oC)
Part C
Basic Solution (pH 10)
Acidic Solution (pH 3)
Neutral Solution (pH 7)
PART A - Observe Normal Catalase Reaction
1. What is happening in your test tube?
2. What do you think would happen if you added more potatoe to the used hydrogen peroxide?
3. What is this used hydrogen peroxide liquid composed of?
4. Is catalase reusable? Explain how you know
PART B - What is the Effect of Temperature on Catalase Activity?
1. What will boiling do to an enzyme?
2. What is the "optimum" temperature for catalase? (This is the temperature at which the
reaction proceeds fastest.)
3. Why did the reaction proceed slowly at 0 deg.C?
4. Why did the reaction not proceed at all at 100 deg.C?
PART C - What is the Effect of pH on Catalase Activity
1. Does there appear to be an "optimum" pH - at what pH? Explain how you would know
2. What is the effect of low or high pH on enzyme activity?
lNTRODUCTlON
.....In general, enzymes are proteins produced by living cells; they act as catalysts in biochemical
reactions. ..A catalyst affects the rate of a chemical reaction. ..One consequence of enzyme activity is
that cells can carry out complex chemical activities at relatively low temperatures. ..In an enzymecatalyzed reaction, the substance to be acted upon (the substrate = S) binds reversibly to the active
site of the enzyme (E). ..One result of this temporary union is a reduction in the energy required to
activate the reaction of the substrate molecule so that the products (P) of the reaction are formed.
.....In summary:
E+S -> ES -> E+P
.....Note that the enzyme is not changed in the reaction and can be recycled to break down additional
substrate molecules. ..Each enzyme is specific for a particular reaction because its amino acid
sequence is unique and causes it to have a unique three-dimensional structure. ..The active site is the
portion of the enzyme that interacts with the substrate. so that any substance that blocks or changes
the shape of the active site affects the activity of the enzyme. ..A description of several ways enzyme
action may be affected follows.
1. Ionic Salt concentration. If the salt concentration is close to zero, the charged amino acid side
chains of the enzyme molecules will attract each other. ..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 will occur, and again the enzyme will precipitate.
..An intermediate salt concentration such as that of human blood (0.9%) or cytoplasm is the
optimum for many enzymes.
2. pH. pH is a logarithmic scale that measures the acidity or H+ concentration in a solution. ..When the
pH is in the range of under 7, a solution is said to be acidic; if the pH is 7, the solution is neutral:
and if the pH is in the range of over 7, the solution is basic. ..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 perform optimally in the neutral pH range and are denatured at
either extremely high or low pH's. ..Some enzymes, such as pepsin, which acts in the human
stomach where the pH is very low, have a low pH optimum.
3. Temperature. Generally, chemical reactions speed up as the temperature is raised. ..As the
temperature increases, more of the reacting molecules have enough kinetic energy to undergo the
reaction. ..Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go
faster with increasing temperature. ..However, if the temperature of an enzyme-catalyzed reaction
is raised still further, a temperature optimum is reached; above this value the kinetic energies of the
enzyme and water molecules are so great that the conformation of the enzyme molecules is
disrupted. ..The positive effect of speeding up the reaction is now more than offset by the negative
effect of changing the conformation of more and more enzyme molecules. ..Many proteins are
denatured by temperatures above 40-50°C (but some are still active at 70-80°C, and a few even
withstand boiling.
4. Activators and Inhibitors. 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. ..These molecules can regulate how fast the 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 its shape or
block it. ..Many well-known poisons such as potassium cyanide and curare are enzyme inhibitors
that interfere with the active site of critical enzymes.
CATALASE
.....Catalase is nearly ubiquitous among organisms that can grow in the presence of oxygen
(air). ..The major function of catalase within cells is to prevent the accumulation of toxic levels
of hydrogen peroxide formed as a by-product of metabolic processes - primarily that of the
electron transport pathway. ..The only exceptions are the "lactic acid bacteria," which cannot
synthesize the fundamental building block porphyrin, and hence do not even possess
cytochromes that would otherwise make the toxic H2O2.
.....Each molecule of catalase has four polypeptide chains, each composed of more than 500
amino acids, and nested within this tetrad are four porphyrin heme groups - very much like the
familiar hemoglobins, cytochromes, chlorphylls and nitrogen-fixing enzymes in legumes.
..(Catalase may also take part in some of the many oxidatic reactions that occur in all cells.)
.....In the absence of catalase, this reaction occurs spontaneously, but VERY slowly. ..Catalase
speeds up the reaction rate many thousands times. In the catalase experiment, a rate for this
reaction will be observed and estimated.
.....Much can be learned about enzymes by studying the kinetics (particularly the changes in
rate) of enzyme-catalyzed reactions. ..For example, 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.
.....If the amount of oxygen formed is measured at regular intervals and this quantity is plotted
on a graph, a curve like the one that follows is obtained.