Biology

ENZYMES
Student Learning Outcomes
At the completion of this exercise you should:
(1) Be able to describe how Benedict’s test can be used to detect the presence of reducing sugars
like Glucose and Fructose.
(2) Be able to describe how enzymes, like Invertase, work on their substrates to produce
products.
(3) Be able to describe the general effect that temperature and pH have on the rate of an
enzyme’s activity.
(4) Be able to describe the question, hypothesis and results obtained in the temperature and pH
enzyme experiments.
(5) Be able to define pH and describe what a pH buffer is.
(6) Be able to design an experiment to test the effect of iodine on the activity of the enzyme
invertase.
I. Preliminary Observations
A. Introduction: General Information about Enzymes
Enzymes are substances which make it possible for organisms to carry out the variety of
metabolic activities necessary for life. Each of the numerous chemical reactions in a living
system is mediated by an enzyme. For example, in the lumen of the mammalian digestive tract,
especially the small intestine, large “food” particles are enzymatically broken down into
molecules small enough to be absorbed into the bloodstream and thus carried to all the cells of
the body tissues. Also, within the individual cells, hundreds of different enzymes catalyze the
breakdown and syntheses of organic molecules in a continuing process which makes possible
cellular maintenance, growth and reproduction.
Enzymes are just one of several categories of proteins. Each enzyme is a protein molecule that
is different in some respect from that of any other kind of enzyme. This uniqueness of structure
allows each type of enzyme to mediate only one specific chemical reaction, or at most a series of
very similar reactions. Enzyme molecules are also known to be very sensitive to their
environments. For example, exposure to high temperatures, chemicals such as strong acids and
other factors often can denature an enzyme’s structure, making it unable to carry out its
function. An enzyme can therefore act only if it is structurally intact, and only within a limited
range of environmental conditions.
Enzymes act as biochemical catalysts. It is thought that their presence reduces the activation
energy necessary to start a chemical reaction. The rate at which the chemical reaction proceeds is
therefore accelerated, and the end products are formed more rapidly. The enzyme molecule itself
remains unchanged during the reaction and does not become a part of the end products.
Therefore, enzymes are reused, as the same enzyme molecule may perform the same function
many times, each time converting a new substrate molecule into the same kind of product
molecule.
B. The Enzyme Invertase: A Representative Example for Today’s Studies
The enzyme invertase has been selected as a representative example for demonstrating the
effects of some environmental factors on enzyme activity. (Enzyme names often have the suffix
“-ase.”) The invertase which we shall use in this experiment was produced by yeast, a
single-celled organism (actually a kind of fungus). In humans, a similar enzyme (called sucrase),
produced by the pancreas, operates in the small intestine.
The specific reaction that invertase catalyzes is the breakdown of the double sugar
(disaccharide) sucrose into the two simple sugars (monosaccharides), glucose and fructose. It
accomplishes this by rupturing the chemical bond that links the simple sugars in the sucrose. The
reaction can occur only in the presence of water: one molecule of water is required to break apart
the disaccharide and, by so doing, completes the structures of the monosaccharides. Breakdown
of molecules with the addition of water is called hydrolysis. This same reaction occurs routinely
in the human small intestine and allows the absorption into the bloodstream of carbohydrates as
monosaccharides.
Question 1. Any enzyme converts substances generally known as substrates into substances
commonly called products. For the reaction shown above, name the substrates and products
below. Then label them on the drawing above.
a. substrate(s):
Replace this text with your answer.
b. product(s):
Replace this text with your answer.
Question 2. If a student mixes some sucrose and invertase solutions in a test tube and allows
some time for reaction, what results would you predict?
Replace this text with your answer.
C. How to Tell If Invertase Has Done Its Job: Benedict’s Test
1. Why Do We Need Benedict’s Test?
Of course, we cannot see the enzyme invertase in solution, as it is colorless. As you may
already know, neither can we see the sugars sucrose, glucose or fructose when they are
dissolved. However, we do wish to measure the rate at which invertase catalyzes the
hydrolysis of sucrose into glucose and fructose. Clever ways to do this might include a
chemical test to reveal the presence or absence of sucrose or a different test for the
products of the reaction. Fortunately, biochemists have developed such a technique,
called Benedict’s test. This test uses a special solution of chemicals – called Benedict’s
reagent a clear, blue solution which gets its color from the dissolved copper compound,
cupric hydroxide Cu(OH)2.
2. Interpretation of Benedict’s Test
When Benedict’s reagent is heated, the solution will change color and develop a
distinctive precipitate, if certain sugars known as reducing sugars have been added. It
has already been established (through experiments) that all monosaccharides, but not all
disaccharides, are reducing sugars. If only a small amount of reducing sugar is present,
the Benedict’s reagent will form a greenish precipitate which may or may not settle out in
the bottom of the test tube. If larger amounts are present, one sees a more yellowish color
with definite precipitation at the bottom of the test tube; with still larger amounts, an
abundance of orange-red-brown precipitate will be apparent. On your tables of results,
you may note the color and use symbols (-, +, ++, +++, etc.) to indicate the relative
amount of reducing sugar revealed by the test.
D. Observing the Benedict’s Test
General Procedure:
1. Turn on your hotplate.
2. Place a small test tube rack in a beaker. Fill the beaker one-third to one-half full of tap water,
and place it on your hot plate. Bring the water to a gentle boil.
3. Throughout this experiment, keep all glassware scrupulously clean. Even trace amounts of
enzyme carried over from one part of the experiment to another may distort the results. Use a
test tube brush, and rinse well because soap can interfere with enzyme activity.
4. Obtain four clean test tubes, and label them 1-4 with a grease pencil.
5. Add two ml (or 20 drops) of “test solution” to a test
tube. (See Table 1 for each of the “test solutions.) Be
sure to read the solution dispenser labels to confirm how
much you are dispensing with each pump
6. To each test tube containing the “test solution,” add two
ml Benedict’s reagent.
7. Hold the sides of the test tube near its top, and then
gently tap the side near the bottom. This will create a small vortex in which the contents are
mixed. Swirl each test tube to mix the contents thoroughly.
8. 8. Place the test tubes in your boiling water bath for three minutes, no longer. Exceeding
this time limit can lead to false positive results
9. Carefully, remove the tubes from the bath, place them in your test tube rack and observe.
(You may use your bare fingers after you test the tops of the test tubes for the temperature, or
you may use test tube clamps if you know how to use them. The tubes shouldn’t get too hot
to handle.)
10. If the solution forms layers, homogenize the solution by gently tapping the side of the tube
near the bottom with the pad of your index finger.
11. Interpret the results for each tube (as described above in Interpretation of Benedict’s Test),
and record the results in Table 1.
E. Using the Benedict’s Test: How can it be used to detect for presence of
monosaccharides (reducing sugars) like Glucose and Fructose?
In order to use Benedict’s test in later experiments, one must first observe the results when
the test is performed on four solutions whose contents are known to us: 1% sucrose, 1%
fructose, 1% glucose and deionized water. Following the directions given in part D
(previous page), perform a Benedict’s test on each of the four known test solutions listed
in Table 1 (below) and record your results.
Table 1: Benedict’s Test on Known Solutions
Test Tube
Number Test Solution
Benedict’s Test
Results:
Color
Benedict’s Test
Results:
Precipitate
(-, +, ++, +++, etc.)
1 1% sucrose
2 1% fructose
3 1% glucose
4 DI* water
* deionized water (water which contains no ions)
Question 3. What was the purpose of testing deionized water?
Replace this text with your answer.
Question 4. A student arrives at the lab on Saturday. S/he wants to verify that the stock invertase
solution left over from Friday’s lab is still “good”. The student mixes some 1% sucrose and some
of the stock enzyme solution in a test tube and lets it stand in the test tube rack for one-half hour.
Then the student performs a Benedict’s test on the test tube contents.
a. What is the correct interpretation of the Benedict’s test if the result is a clear, blue
solution? Is the enzyme active or not?
Replace this text with your answer.
b. What is the correct interpretation if Benedict’s test shows a bright orange precipitate? Is
the stock enzyme still “good”?
Replace this text with your answer.
II. Factors Affecting Enzyme Activity
A. Experiment 1: Effect of Temperature on Invertase Activity
We all know that living systems are extremely sensitive to environmental temperature. Upon
reflection, it may seem likely to you that even the individual chemical reactions of living
cells could possess such sensitivity. To investigate this possibility for enzyme-mediated
reactions, we shall set up a controlled experiment to test the effects of a few selected
temperatures on invertase activity. While it is possible for us to test the enzyme’s response to
a wide spectrum of temperatures. today we have selected just four for observation:
0o C (freezing point of water)
25° C (usual room temperature in our laboratory)
40° C (slightly higher than normal human body temperature–37°C)
100° C (boiling point of water)
Question 5. Based on what you already know about living things, in what general range of
temperature do you expect enzyme molecules to function best?
Question 6. Now we are ready to start writing a hypothesis.
a. What is the dependent variable?
Replace this text with your answer.
b. What is the independent variable?
Replace this text with your answer.
c. What will be the 4 “groups?”
Replace this text with your answer.
d. State your answer as a hypothesis for this part of the exercise in an If…, and …, then …
format.
Replace this text with your answer.
Procedure:
1. Prepare enzyme tubes:
a. Label four clean test tubes respectively as follows: “0°C enzyme” “25°C enzyme,”
“40°C enzyme” and “100°C enzyme.”
b. To each of the “enzyme” tubes, add 2.0 mL stock enzyme solution and 2.0 mL pH 4.4
buffer solution.
c. Homogenize the solutions by tapping the bottom of your test tube as you did in the
Benedict’s test.
2. Prepare substrate tubes:
a. Obtain four more clean test tubes, and label as follows: “0°C substrate,” “25°C
substrate,” “40°C substrate” and “100°C substrate.”
b. To each of the “substrate” tubes, add 2.0 mL 1% sucrose solution.
3. Place all eight test tubes in their marked temperature conditions as follows:
a. Place both “0°C” tubes in the ice bath with ice slurry on the side bench near the sink.
b. Place both “25°C” tubes in your test tube rack on your lab bench.
c. Place both “40°C” tubes in the warm water bath near the sink.
d. Place both “100°C” tubes in the boiling water bath on your bench.
4. Check the temperatures of the 0°C and 40°C baths to confirm their temperatures. If there are
differences in the actual temperatures, write down the actual temperatures next to letters a-d
in step 3 above.
5. Allow ten minutes for the contents of the tubes to reach the actual temperatures of their
respective environments.
6. After the ten-minute equilibration period, mix the contents of the
tubes as follows:
a. Pour the contents of each “substrate” tube into the ”enzyme”
tube at the same temperature.
b. Mix the solutions by tapping the test tube bottoms with the
pad of your index finger. Then return each “enzyme” tube
to its marked temperature environment. (The empty
“substrate” tubes may now be washed for later use.)
7. At this point, allow ten minutes more for the enzyme-substrate
reaction to proceed at the various temperatures.
8. Use Benedict’s Test to assess invertase’s performance.
a. While you are waiting for completion of step 6 (above), thoroughly wash and number
four more test tubes in preparation for performing Benedict’s test. Remember to label
the tubes.
b. Add 2.0 mL Benedict’s solution to each tube.
c. Obtain four clean pipettes.
d. After you have allowed 10 minutes for the enzyme–substrate reactions (as directed in
part “6” above), gather the four reaction tubes from the 0°C, 25°C, 40°C and 100°C
water baths and place them on the table next to your four labeled tubes containing
Benedict’s solution. Immediately proceed to the following steps.
e. Using a clean pipette, transfer 20 drops of your enzyme-substrate reaction mixture
from the tube labelled “0° C.” to the tube containing 2 mL Benedict’s solution, labelled
“0° C.”
f. Using another clean Pipette each time, repeat step “e” for the “25° C”, “40° C” .and
“100° C” tubes.
g. Now place the four Benedict’s test tubes in your boiling water bath for exactly three
minutes. Use a clamp to remove them to your test tube rack.
h. Record your results in Table 2.
Table 2: Invertase Activity at Different Temperatures
Test Tube
Number
Temperature of
Test Solution
Benedict’s Test
Results:
Color
Benedict’s Test
Results:
Precipitate
(-, +, ++, +++, etc.)
1 0 o C
2 25 o C
3 40 o C
4 100 o C
Question 6. a. At which temperature was invertase most active?
Replace this text with your answer.
b. At which temperature was invertase least active?
Replace this text with your answer.
c. Describe how you arrived at your decision:
Replace this text with your answer.
Question 7. Recall Exercise 3: “Diffusion and Osmosis”, where you observed the India ink dye.
What happened to the molecular motion as you increased the light intensity (heat)? In that
exercise, you learned the relationship between heat and molecular motion.
a. State the relationship:
Replace this text with your answer.
b. Referring to Table 2, identify which of your results can be explained by this relationship.
Explain your reasoning.
Replace this text with your answer.
c. Which part of your Table 2 data are not consistent with the relationship between heat and
molecular motion?
Replace this text with your answer.
d. What do you know about enzymes (and proteins in general) which can account for your
results at 100°C? (You may need to consult your textbook’s discussion of protein
molecules to answer this question.) This should include the following concepts:
1) Structure/function/denaturation
2) Bonds – types of bonds affected
Replace this text with your answer.
Clean up
1. Drain the test tubes of solutions down the sink.
2. Rinse and wash the test tubes with soap, then rinse with plenty of tap water.
3. You will be using the test tubes and other materials for the next part.
B. Experiment 2: Effect of pH on Invertase Activity
The pH of a solution tells us its relative acidity or basicity. (“alkaline” is another word for
“basic.”) It is well known that most living things cannot survive pH extremes, and generally
thrive in environments whose pH values are close to neutral. For example, the cells of your
body are living in a fluid whose pH averages about 7.4. As added “protection” against pH
extremes, body fluids contain solutes called buffers, whose chemical nature causes the fluid
to resist changes in pH. Many cellular waste products are acidic and could decrease the
body’s pH if the buffers were not present.
If living cells are sensitive to pH, perhaps enzyme-mediated reactions also possess such
sensitivity. Today we shall observe the activity of the enzyme invertase in four buffered
solutions* at the following pHs:
pH 1.0 pH 4.4 pH 8.0 pH 12.0
*Note: the word “buffer” is used to describe pH solutions whose chemical nature causes
them to resist changes in pH.
Question 9. List the pH’s of the above buffered solutions in order from most acidic to most
basic:
Most acidic → → Most basic
pH:
Question 10. What pH is called “neutral”? (Refer to your textbook.) _______
Question 11. What is the pH of pure water? (Refer to your textbook.) _______
Question 12. Based on what you already know about living things, in what general range of pH
do you expect enzyme molecules to function best? ________
Question 13. Now let’s write a hypothesis.
a. What is the dependent variable?
Replace this text with your answer.
b. What is the independent variable?
Replace this text with your answer.
c. What are the 4 groups in this situation?
Replace this text with your answer.
d. State your answer as a hypothesis for this part of the exercise in an If…, and …, then …
format.
Replace this text with your answer.
Procedure:
(Caution: pH 1.0 and 12.0 can burn skin or clothing. If spills occur–even a drop on skin or
clothes–flush with plenty of water and call your instructor.)
1. Set up enzyme reactions in four selected pH buffer solutions. Be sure to mix the solutions in
the order indicated:
a. Label 4 clean test tubes respectively as follows: “pH 1.0”, “pH 4.4”, “pH” 8.0″ and “pH
12.0.”
b. Add 2.0 mL stock enzyme solution to each tube.
c. Add 4.0 ml of the proper pH buffer solution to each tube. Use the marked laboratory
stock pH buffer solutions.
d. Add 2.0 mL 1% sucrose solutions to each tube.
e. Homogenize the solutions by tapping the bottom of your test tube as you did in the
Benedict’s test.
f. Allow 10 minutes for the enzyme-substrate reactions to proceed.
2. Use Benedict’s test to assess invertase’s performance.
a. While you are waiting for completion of step 1e (above), wash and number four test
tubes in preparation for performing Benedict’s test. Remember to label the tubes (pH
1.0, pH 4.4, pH 8.0, pH 12.0).
b. Add 2.0 ml Benedict’s solution to each tube.
c. Obtain four clean pipettes.
d. After you have allowed 10 minutes for the enzyme substrate reactions, as directed in
part 1e (above), immediately
proceed with the following steps.
e. Using a clean pipette, transfer 20
drops of your enzyme-substrate
reaction mixture from the tube
labeled “pH 1.0” to the tube
containing 2 ml Benedict’s solution,
labeled “pH 1.0.”
f. Using another clean pipette each time, repeat step “e” (just above) for the “pH 4.4,” “pH
8.0” and “pH 12.0” tubes.
g. Now place the four Benedict’s test tubes in your boiling water bath for exactly three
minutes. Use your bare fingers after testing for temperature or a test tube clamp to
remove them to your test tube rack.
h. Record your results in Table 3.
TABLE 3: INVERTASE ACTIVITY AT DIFFERENT pHs
Test Tube
Number
pH of Test
Solution
Benedict’s Test
Results:
Color
Benedict’s Test
Results:
Precipitate
(-, +, ++, +++, etc.)
1 1.0
2 4.4
3 8.0
4 12.0
Question 14. At which pH was invertase most active?
At which pH was invertase least active?
Question 15. Thought question: Assuming yeast cells (from which your invertase was extracted)
could survive pH 1.0 or pH 12.0, do your results suggest that they could obtain their energy from
sucrose? Explain.
Replace this text with your answer.
Question 16. In the body of an organism such as yourself, do you expect all enzymes to operate
effectively in the same pH range? For example: compare the pH of the stomach, where the
digestive enzyme pepsin works, and the pH of blood, where other enzymes are found.
Replace this text with your answer.
Question 17. What would you guess to be the normal pH inside of human cells, where there are
many different enzymes?
Replace this text with your answer.
Clean up
Your instructor may choose to continue to Part C. If so, clean up after then.
1. Drain the test tubes of solutions down the sink.
2. Rinse and wash the test tubes with soap, then rinse with plenty of tap water.
3. Place clean test tubes in the bucket in the sink.
4. Used pipettes go in trash.
5. Turn off hot plates.
6. Wipe down your lab bench with paper towels yellow cleaning solution.
7. Push in your chair.
C. Experiment 3: Effect of a Possible Chemical Inhibitor (Iodine) on Invertase Activity
In addition to various other environmental conditions, certain chemicals are known to inhibit
the activities of many enzymes. In this exercise, your assignment is to determine whether an
iodine solution inhibits the activity of invertase.
Question 17.
a. Name the enzyme to be used in your experiment:
b. Name the substrate to be used in your experiment:
Question 18. What is the dependent variable of this experiment?
Replace this text with your answer.
Question 19. What is the independent (experimental) variable of this experiment?
Replace this text with your answer.
Question 20. State your hypothesis:
Replace this text with your answer.
Procedure:
This time, you will design and execute your own experiment. However, since you will be using
the same enzyme and substrate used in the temperature and pH experiments, many parts of
your design will be the same.
Remember that “good” science requires the use of a control. All test tubes should have contents
as identical as possible, except for your experimental variable, iodine.
Question 21. Describe what is meant by the phrase “controlled experiment.”
Replace this text with your answer.
Now, design your controlled experiment for iodine. You will be led through the process as
you answer Questions 17-22 below. When asked, write the quantities of materials you will place
in the test tubes (“treatment levels”) in Table 4 on the next page. You have the option of using
either two or three test tubes. Before you start your experiment, consider the following
questions, and then check with your instructor.
Question 22. Your instructor suggests that the total volumes in all tubes should be identical.
Why?
Replace this text with your answer.
Question 23. What is the solvent in all of your solutions? How can the solvent be useful in
designing your experiment? (Hint: See Question 19.)
Replace this text with your answer.
Question 24. Some students decided to use the same pH buffer solution in all of their tubes.
Why would this be useful? (Which buffer solution will you use? Why? (Hint: Review your
results in Table 2.)
Replace this text with your answer.
Question 25. What temperature will you use for your experiment? Why? (Hint: Review your
results in Table 2.)
Replace this text with your answer.
Question 26. Students have suggested that the enzyme be “pretreated” with the iodine before
adding the substrate. Why would this be useful? (Hint: If you added the substrate to the enzyme
first, think what might be happening while you were on your way over to the iodine bottle.)
Replace this text with your answer.
Question 27. How much time will you allow for the enzyme-substrate interaction before
performing Benedict’s test?
Replace this text with your answer.
Table 4: Experimental Design for
Effect of Iodine on Invertase Activity
(NOTE: THE ANSWERS TO QUESTIONS 14-15 WILL HELP YOU COMPLETE THIS TABLE)
Solutions Tube # 1
Contents
Tube # 2
Contents
Tube # 3
Contents
ml. (or “drops”) of iodine
ml. enzyme name:
ml. substrate name:
ml. buffer pH:
ml. DI water
Total Volume
Question 28. Results: execute your experiment as planned, then perform Benedict’s test and
enter your data in the table below.
Tube # 1 Tube # 2 Tube # 3
Benedict’s test results
Clean up
1. Drain the test tubes of solutions down the sink.
2. Rinse and wash the test tubes with soap, then rinse with plenty of tap water.
3. Place clean test tubes in the bucket.
4. Used pipettes go in trash.
5. Turn off hot plates.
Question 29. Iodine experiment conclusions:
a. Was your hypothesis supported, refuted or were your results inconclusive?
Replace this text with your answer.
b. Describe how analysis of your results led to your conclusion:
Replace this text with your answer.
Question 30. You may be aware that iodine has been used as an antiseptic. What is an
antiseptic? (Look up the definition.)
Replace this text with your answer.
Question 31. What have you learned about iodine to help you understand its use as an
antiseptic?
Replace this text with your answer.

Guidance for Enzyme Lab Report
This lab report will focus on your evaluation of how temperature and pH affect the rate of enzyme
activity.
Introduction / Purpose (5 points)
Why did we do this lab?
Are there any important concepts or explanations that are relevant to the reader’s understanding of
the purpose and background of the lab?
What is invertase? What chemical reaction does it catalyze? This can be a diagram.
What were your hypotheses for each of the experimental procedures we conducted?
What is Benedict’s test? How and why does it work? Why did we use it? What are reducing
sugars?
Materials & Methods (5 points)
Follow the directions provided above regarding the level of detail to include here.
Convey the experimental design using a labeled diagram or image, if desired.
Results & Data (5 points)
Include any tables from the lab containing the data you collected
Include a written statement of the results of every single experiment you conducted.
Discussion & Conclusion (10 points)
Scientifically interpret your results for every experiment you conducted.
This means every temperature, and every pH.
Can you relate your results to Brownian motion or diffusion?
Can you relate your results to the effect of pH on secondary structure?
What does it actually mean when an enzyme is denatured by heat?
Say you obtained a negative result from Benedict’s test when you carried out the chemical reaction
at the highest pH.
Why did you observe these results? You must provide an explanation that is based on your
knowledge of enzymes, how they function, etc.
You may not completely understand why the invertase didn’t function in those conditions…it is your
job to learn this.
You may have to reference your textbook or another reference to gain sufficient understanding of
your experimental outcomes! 🙂

1. State and interpret your results for the enzyme experiment at each of the four temperatures.
Did your results make sense? Why or why not?
What did your results tell you about enzyme activity under each temperature condition?
Why did invertase behave the way it did in each condition?
What conclusions can you draw about the activity of invertase in biological organisms?
Use your textbook and other resources to better understand and explain your results.
ANY INFORMATION YOU DERIVE FROM ANOTHER SOURCE MUST BE CITED.
2. State and interpret your results for the enzyme experiment at each of the four pH levels.
Did your results make sense? Why or why not?
What did your results tell you about enzyme activity under each condition?
Why did invertase behave the way it did in each condition?
What conclusions can you draw about the activity of invertase in biological organisms?
Use your textbook and other resources to better understand and explain your results.
ANY INFORMATION YOU DERIVE FROM ANOTHER SOURCE MUST BE CITED.
3. Create a diagram or flow chart showing the experimental procedure for the enzyme temperature
and pH experiments.
4. Explain the chemistry behind Benedict’s reagent. How is Benedict’s reagent affected by the
presence of reducing sugars? Chemically speaking, why does this change take place?
5. Given your answers to question 4, why do we use Benedict’s reagent to test for the activity of
invertase?
6. Draw the chemical equation for the breakdown of sucrose.