Laboratory 2, page

LABORATORY 2: ENZYME ACTION

A catalyst speeds up a chemical reaction by lowering the activation energy required (textbook Chapter 5
and Figure 5.7). 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. Sometimes
an enzyme incorporates an important nonprotein component into its structure. If this component is attached
to the enzyme's protein covalently, it is a prosthetic group. If it is held more loosely by other types of
bonds, it is a cofactor. Examples of cofactors are inorganic ions and organic coenzymes. You are already
familiar with many coenzymes that are required in the human diet and are known as vitamins.

In an enzyme-catalyzed reaction, the substance to be acted upon, or substrate, binds to the active site,
or business end, of the enzyme. The enzyme and substrate are held together in an enzyme-substrate
complex by hydrophobic interactions, 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 into solution 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 over and over. One enzyme molecule can carry out
thousands of reaction cycles every minute.

Each enzyme is specific for a certain reaction because its unique amino acid sequence causes it to have a
unique three-dimensional structure. 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. If there is a prosthetic group on the
enzyme, it will form part of the active site. 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, 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 or most efficient
enzyme activity.

1. Salt concentration. If the salt concentration is very low or zero, the charged amino acid side chains of
the enzyme molecules will stick together. The enzyme will denature and form 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 at that of blood (0.9%) or cytoplasm is the optimum for most enzymes.

2. pH. pH is a logarithmic scale that measures the acidity or H + concentration in a solution. The scale
runs from 0 to 14 with 0 being highest in acidity and 14 lowest. When the pH is in the range of 0-7, a
solution is said to be acidic; if the pH is in the range of 7-14, the solution is basic. Amino acids contain
side chains such as carboxyl (COOH) or amino (NH 3+) groups 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.
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Figure 1: Side chains of amino acids

Some enzymes, such as those that act in the human stomach where the pH is very low, will have an
appropriately low pH optimum. A buffer is a compound that acts like a sponge to pick up any extra H + or
OH- ions so that a constant pH is maintained. The buffer molecules (B) must be in two forms to do this:
Different buffers are designed to keep the pH at various pH levels.

Figure 2: Buffer molecules

3. Temperature. All chemical reactions speed up 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 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 40-50 oC, but some are still active at
70-80oC, and a few even withstand being boiled.

4. Modulator molecules. Many small 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
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rate it is an inhibitor. The cell can use these modulators to regulate how fast the enzyme acts. Any
substance that tends to unfold the enzyme, such as an organic solvent or detergent, will also 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 that interfere with the active site of a critical enzyme.

I. Study Objectives and Questions:

1. Give the class of macromolecules to which peroxidase belongs and the monomers of which it
is composed.
2. Name the substrates and products of the peroxidase catalyzed reaction.
3. Explain the role of guaiacol in this experiment.
4. Define enzyme, activation energy, active site, pH, and denaturation.
5. Distinguish between oxidation/reduction, activation energy/catalysis, substrate/product,
modulator/prosthetic group, and hydrogen peroxide/peroxidase.
6. Define the term optimum with respect to peroxidase activity.
7. Describe how temperature, pH, enzyme concentration, and substrate concentration affect the
reaction rate.
8. Explain why peroxidase is a necessary enzyme for all aerobic or oxygen-utilizing cells.

TERMS YOU SHOULD BE ABLE TO DEFINE:

chemical reactions proteins enzyme-substrate interactions

catalyst enzymes pH

II. Laboratory Exercise: Measuring the activity of turnip peroxidase

In this experiment you will study the enzyme peroxidase from turnips. Peroxidases are enzymes that are
widely distributed in plant and animal cells and catalyze the oxidation of organic compounds by hydrogen
peroxide (H2O2) as follows:

Figure3: The peroxidase reaction

QUESTION #1 What are the substrates in this reaction? What are the products?

Any cell using molecular oxygen in its metabolism will produce small amounts of H 2O2 as a highly toxic
byproduct. A peroxide has a very reactive _O_O_ structure, so it is critical that it be quickly removed by
enzymes such as peroxidase before it can do damage to the cell. In order to follow the reaction as it
proceeds, you will be using a substrate, the reduced, colorless form of a substance called guaiacol. It is an
 Laboratory 2, page

organic chemical produced by the guaiac tree of Central America and it corresponds to H _R_O_H in the
equation. In the reaction, guaiacol will donate hydrogens and thereby become oxidized. It has a brown
color in its oxidized form and you will be able to measure the amount of oxidized guaiacol produced by
determining the intensity of the color in the spectrophotometer at a wavelength of 470 nm.

Figure 4: The guaiacol reaction

In this reaction, H2O2 is reduced to water by giving up an atom of oxygen as guaiacol gives up 8 of its
hydrogens. The oxygen and hydrogen combine to form more water. The decomposition of H 2O2 is thus a
good example of an oxidation-reduction reaction.

III. INTRODUCTION TO THE SPECTROPHOTOMETER

A spectrophotometer is an instrument that measures the amount of light of a selected wavelength that
passes through colored or turbid (cloudy) solutions.

Figure 1: The spectrophotometer

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The spectrophotometer is usually used to achieve one of the following goals:

a) To determine the concentration of a solution. This is possible because concentration is related to

the amount of light absorbed or scattered by the solution.

b) To determine the identity of an unknown substance(s) in the solution by observing which

wavelengths of light are absorbed by the solution.

INSTRUCTIONS FOR OPERATING

A. For Initial Readings:

1. Turn the instrument on by rotating the Power Switch/Zero Control (lower left knob)
clockwise. Allow 15 minutes for warm-up.

2. Set zero: Close the cover of the Sample Well and adjust the meter needle to ∞ on the
Absorbance scale (Optical Density) by turning the Power Switch/Zero Control knob.

3. Set to desired wavelength the Wavelength Control knob. For the turnip
peroxidase/guaiacol experiment, set the wavelength to 470nm.
4. Insert a cuvet containing your "Blank" solution in to the Sample Holder and close the
lid.

5. Set full scale, i.e., wait until the needle stops moving and then adjust the meter reading to
0 Absorbance, i.e., 100% Transmittance (O.D.), with the 100% T Control knob.

6. Insert unknown: Remove the Blank, insert the tube containing your unknown, and close
the Sample Holder lid.

7. Read % Transmittance or Absorbance (check your manual) of your unknowns.

B. For Further Readings:

1. At same wavelength:

Repeat step 6. From time to time, repeat steps 4 and 5.

2. At different wavelengths:

Repeat step 5 each time you change the wavelength, then repeat step 6.

Repeat step 4 from time to time (zeroing the instrument with your blank)

NOTE: Successful use of your spectrophotometer depends upon the consistent use of
correct laboratory techniques. To minimize problems, follow these simple guidelines:

1. Keep all solutions free of bubbles.

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2. Make sure that all Spec 20 tubes are at least half full and that the index mark on the test
tube aligns with the mark on the sample chamber.

3. During extended operation at a fixed wavelength, check from time to time for 100%T by
zeroing the instrument with your blank.

4. Use clean Spec 20 tubes and do not touch the test tubes below the index mark. Wipe the
tubes with Kim wipes before taking readings.

IV. Experimental Procedure:

Turnip Extract will be provided by your instructor. The turnip extract contains the enzyme peroxidase.
The activity of the turnip extract will vary from day to day, depending on the size and age of the turnip and
the extent of blending. You should adjust the turnip suspension to be more dilute or more concentrated so
that your absorbance for the base line is in the range 0.1-0.2.

Kinetics of the Peroxidase Reaction. Pipets will be used to measure accurately the solutions used in
this experiment. Your instructor will demonstrate how to use a pipet correctly.

Be sure to USE A DIFFERENT PIPET FOR EACH SOLUTION so that the reagents are NOT
RUINED BY CROSS-CONTAMINATION.

Label the pipets with tape or a marking pencil so each one can be reused with the proper solution.
Obtain two spectrophotometer tubes and label them C (control) and R (reaction)

Obtain three test tubes and label them #1, #2, and #3. #1 will contain a control reaction without H 2O2. The
contents of #2 (substrate) and #3 (enzyme) will be mixed to start the reaction.

Set up the three tubes as follows, and make a record of these additions in Table 1 (Run 1, base line) at the
end of this topic.

Tube #1 (control tube without H 2 O 2 ): Add 0.1 ml of guaiacol, 1.0 ml of turnip extract, and 8.9 ml
of distilled water; mix well.

Tube #2 (substrate):Add 0.1 ml of guaiacol, 0.2 ml of 0.1% H 2O2, and 4.7 ml of distilled water.

Tube #3 (enzyme): Add 1.0 ml of turnip extract and 4.0 ml of distilled water.
 Laboratory 2, page

Tube #1 Tube #2 Tube #3

Run 1
(Base line)

Run 2

Run 3

Run 4

Run 5

Run 6

Run 7

Table 1

Adjust the Spectronic 20 to zero absorbance using tube C filled with solution from tube #1 and following
steps 1-5 in the instructions for its operation. The wavelength should be set at 470nm.

You have now set up the instrument so that any difference in the meter reading with a change in sample will
reflect a difference in oxidized guaiacol concentration.

Obtain a stopwatch, and be sure that you understand how to use it correctly.
Prepare the sample: have ready spectrophotometer tube R, tissue, and tubes #2 and #3 filled with the
solutions given above.

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------------
You will have 30 sec to mix the contents of tubes #2 and #3, pour the contents into the clean
spectrophotometer tube, wipe the tube, and take your first reading.
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------------

When you are completely ready, mix the contents of tubes #2 and #3, pour the contents back and forth two
times, and then pour quickly into tube R; start the stopwatch when the tubes are mixed ( t = 0 when the
tubes are mixed.)
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Wipe the outside of the tube and place it in the spectrophotometer (wavelength set at 470 nm).

Take your first reading 30 sec or as soon as possible after the tubes were mixed ( t = 30 sec).

Continue to read the absorbance every 30 second for 3 min.

Record the readings in Table 2, and graph the absorbance versus time in Figure 1.

Time Run 1
(run) (Base line)

30

60

90

120

150

180

Table 2

This curve will represent the base line of enzyme activity with which the enzyme activity under varying
conditions will be compared. In the following experiments, you will vary one condition at a time and
compare the results with the base line. Be sure you have already recorded the setup for your base line
experiment under run 1 in Table 1. Check your graph with your instructor before proceeding with the rest of
the experiment.

Since this exercise contains too many projects to be performed by one pair of students, the instructor will
specify how the remaining work will be divided up. Use the experimental setup space in Table 1 to record
your experiment number and what you put into your tubes; record your data in Table 2 and graph your
results. An extra page for graphing is included, in case you need more space.

NOTE: Your GSI will assign you one of the following projects.

1. Effect of Enzyme Concentration

What happens when you use:

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(a) Twice the amount of enzyme

Tube #1. Add 0.1 ml of guaiacol, 2.0 ml of turnip extract, and 7.9 ml of distilled
water.
Tube #2. Add 0.1 ml of guaiacol, 0.2 ml of 0.1% H2O2, and 4.7 ml distilled water.
Tube #3. Add 2.0 ml of turnip extract and 3.0 ml of distilled water.

(b) Half the amount of enzyme

Tube #1. Add 0.1 ml of guaiacol, 0.5 ml of turnip extract, and 9.4 ml of distilled
water.
Tube #2. Add 0.1 ml of guaiacol, 0.2 ml of 0.1% H2O2, and 4.7 ml of distilled water.
Tube #3. Add 0.5 ml of turnip extract and 4.5 ml of distilled water.

Notice that tube #1 always contains 10 ml and tubes #2 and #3 together total 10 ml.

QUESTION #2 Why is this important?

Repeat the procedure for preparing the sample, read it, record the data, and graph your results.

QUESTION #3 How does changing the concentration of enzyme affect the rate of the reaction?

2. Effect of varying the Substrate Concentration

Design and carry out an experiment to determine whether varying the concentration of the
substrate H2O2 affects the rate of reaction. If substrate were present in excess, there would be
little or no effect.

QUESTION #4 What would happen if the concentration of this substrate were increased further
and further?

3. Effect of Temperature

QUESTION #5 What is the effect of temperature on enzyme activity?

Set up an experiment to show the difference in activity found between 0C and 60C. Use the
conditions of your base experiment, but run the reaction in the water baths at the different
temperatures that are available to you, such as 4C, 15C, room temperature (about 22C), 30C,
37C, and 55C. Record the exact temperature for each run.

Place tubes #2 and #3 in the water bath to equilibrate for 5 min before mixing.

Read the absorbance immediately after mixing; then return tube R to the water bath and wait.

Read the absorbance again at 3 min. Graph your two data points for each temperature.
 Laboratory 2, page

Next, set up another run but heat tube #3 in a boiling water bath at 100C for 10
min, and test its activity as in Run 1.
QUESTION #6 Is the boiled enzyme active? Explain what happened during boiling.

QUESTION #7 At what temperature in the range 0-100C do you see maximal activity? This
temperature is called the enzyme's temperature _________________________

4. Effect of pH

Measure the effect of pH by using the base line conditions but substituting buffers at pH values
between 4 and 9 (4.0, 5.5, 7, and 8.5) for distilled water in tube #3. Compare each pH rate with the
others. (You cannot compare the base line and buffer results, because buffer itself will change the
rate.)

QUESTION #8 At which pH is the enzyme most effective? Why is this important biologically?

5. Effects of Inhibitors

Hydroxylamine is a small molecule whose structure is similar enough to that of H 2O2 that it
attaches to the iron atom that is part of the peroxidase enzyme. Its inhibition is reversible and
competitive. To test how this substance affects enzyme activity, in tube #3 substitute 3.0 ml of
distilled water and 1.0 ml of 10% hydroxylamine. Mix this tube and wait 1 min. Then measure the
activity of the peroxidase as usual.

QUESTION #9 Is the peroxidase still active? How could you restore activity in the presence of
this inhibitor?

QUESTION #10 Explain at the molecular level the action of hydroxylamine on the enzyme's
activity.

When you are finished with your work, wash and thoroughly rinse your glassware, return the reagents, and
discard any debris left from your experiment. Rinse thoroughly but DO NOT BRUSH your
spectrophotometer tubes.