Background

One of the qualities that distinguishes living things from non-living things is that living things have the ability to carry out chemical reactions that are crucial for survival. Even single-celled
organisms are able to perform hundreds of chemical reactions within their cell wall. Imagine the countless number of reactions that take place in a large organism such as a human! None of these
reactions are possible without enzymes.
Enzymes are biological catalysts or reaction assistants. Enzymes are made up of various types of proteins that work together to drive a chemical reaction. Enzymes can get a reaction underway or
can speed it up. For example, in the presence of certain enzymes, substrates (the chemical reactants) can be transformed into usable products at the rate of millions of times per second! In the
absence of the enzymes, however, the reactants could take years to be converted into usable products, if at all. The work of enzymes is crucial to the existence of life on Earth.
Enzymes are globular proteins: they have a specific 3D shape that is determined by the electrostatic charges of their constituents attracting and repelling one another. The 3D shape of a protein is
critical to its function. Enzyme activity is described by a lock and key model, also known as the induced fit model.
Enzymes have active sites where they come into contact with specific substrates. Once a substrate has come into contact with the active site of an enzyme, it is manipulated by the enzyme into
the final product. When the process is complete, the enzyme releases the product and is ready to begin the process with new substrates. Enzymes are reusable and therefore always recycled.

The environment of an enzyme also has an effect on its 3D shape. For example, pH affects the shape of the enzyme's active site. In the presence of excess H
+
or OH
-
ions, the active site on the
enzyme becomes progressively distorted; this decreases the enzyme's activity until it can no longer function as a catalyst. This process is called denaturation of the enzyme.
Temperature also affects the activity of enzymes. Chemical reactions accelerate as temperature increases, so, in general, catalysis will increase at higher temperatures. However, each enzyme
has an optimum temperature point beyond which the enzyme's functional 3D shape is lost and catalysis decreases. Boiling temperatures will denature most enzymes by stretching the molecular
bonds present in the enzyme beyond repair.
Many enzymes that are present in our bodies help us break down our food. Digestive enzymes are secreted all along the digestive track including the mouth (saliva), stomach, small intestine, and
large intestine. Each stop along the digestive track has its own physiological environment and the enzymes in each environment must be able to perform certain functions. The enzyme, amylase,
breaks down starches into simpler sugars. Another enzyme, lipase, breaks down fats into simpler molecules. Proteases break down proteins into simpler molecules called peptides.
Enzymes make use of the chemical process of hydrolysis, in which a larger molecule is cleaved into two parts by the addition of a molecule of water. Thus, if a compound is represented by the
formula AB in which A and B are atoms or molecules, and water is represented by the formula HOH, the hydrolysis reaction may be represented by the reversible chemical equation:
AB + HOH AH + BOH
As mentioned above, amylase breaks down starches into simpler sugars. Amylase catalyzes the breakdown of starch by cutting off the disaccharide maltose (two glucose molecules linked
together). As the reaction progresses, the starch disappears and the sugar content (maltose) increases.
Experiments
In the experiments that follow, you will perform some tests to learn about enzyme activity.
These experiments examine the effects that environmental pH and temperature have on the rate of amylase digestion of starch. This will be observed by using iodine to test for the presence of
starch. Recall that iodine interacts with starch to form a dark blue-black color. As amylase breaks down starch, less and less starch is present, so the color of the iodine test solution becomes
lighter and lighter until only the amber color of iodine ions will be seen.
In order to stop the reactions at a specific time, we take advantage of the fact that enzyme activity is affected strongly by pH. When a strong acid (HCl) is added to an amylase solution, the pH falls
below the functional pH of the enzyme and the reaction stops. In the case of amylase, the optimum pH is approximately 7 (neutral pH).


Procedures

One way to test the activity of amylase is to find the rate at which it breaks down
a known quantity of starch. You may remember testing for the presence of starch
with a solution of iodine (I
2
KI). Starch chains wrap around the tri-iodide ion in a
coil. It is this shape, shown below, that produces the deep purple color.

If we add amylase to the solution, it breaks down the starch until there are no
chains long enough to wrap around the tri-iodide, and the color of the solution
returns to the yellow tint of plain iodine ions.
Experiment 1: Calibrating the Spectrophotometer for
Starch-Iodine Measurements
A spectrophotometer measures the absorbance of light as it passes through a
solution. In order to use it to make quantitative measurements, a calibration
curve must be generated for the specific makeup of the solution being tested.
For this set of experiments, we'll use the iodine test for starch, which yields a
solution with a deep blue-black color. While the white light shines through the
solution, we see the blue-black color because its opposite color, yellow-orange,
is being absorbed from the light's full spectrum of colors.
We therefore need to find the relationship between the absorbance of
yellow/orange light by the tri-iodide ions in starch as a function of the amount of
starch in solution. This is done by measuring the absorbance of three solutions of
known starch concentration and constructing a calibration curve from this data.
The calibration curve will be roughly linear and can be represented by a best-fit
line which mathematically represents the relationship between concentration and
absorbance.
y = m x +
b
Where m is the slope and b is the y-intercept
If the experiment is performed properly, your y intercept will be zero and you'll
only need to use the slope to determine concentration.
1. Place three cuvettes from the Containers shelf onto the workbench.
2. Place an Erlenmeyer flask onto the workbench.
3. Add 1 mL of the 2% Starch Solution from the Materials shelf to the Erlenmeyer
flask. Note: For the calculations you will perform in the assignments you
will assume that, because this solution is very dilute, its density is the
same as pure water: 1 g/mL. Therefore, there are 20 mg of starch per mL
of the 2% solution.
4. Add 99 mL of water to the Erlenmeyer flask to obtain a dilute solution that is
1/100th the concentration of the original 2% Starch Solution on the shelf.

5. Using the diluted starch in the flask and the Iodine solution found on the
Materials shelf, fill the three cuvettes as follows:
6. Exp. 1 Cuvette Set-up
7. Cuvette Name 8. Starch Solution (mL) 9. Iodine (mL)
10. 1 11. 4 12. 1
13. 2 14. 2 15. 3
16. 3 17. 1 18. 4
19.
20. Place a spectrophotometer from the Instruments shelf onto the
workbench. Using the control panel:
Select the Vis button.
Set the wavelength to 620 nm by clicking the < and > buttons.
Select the A button to set Absorbance.
21. Move cuvette 1 into the spectrophotometer's loading well. The digital
display will show the absorbance once the lid closes.
22. Record the cuvette's number, contents, and absorbance in your lab notes.

23. Repeat this procedure to identify the absorbance of the remaining two
cuvettes. Remember to save your notes. You will use this data to
construct the calibration curve for absorbance vs. starch concentration in
the lab assignments.
24. Discard the cuvettes in the recycling bin.
Experiment 2: The Effect of pH on Amylase Enzyme
Activity
Part 1: Measuring the Initial Starch Absorbance
1. Place three clean cuvettes from the Containers shelf onto the workbench.
2. Using items found on the Materials shelf, fill the three cuvettes as follows:
3.
4. Exp. 2 Cuvette Set-up
5. Cuvet
t
e

N
a
m
e
6. Wa
t
e
r

(
m
L
7. Starch
Soluti
on
(mL)
8. Hydrochloric
Acid
(mL)
9. Iodine
(m
L)
)
10. 1 11. 7 12. 3 13. 14.
15. 2 16. 17. 18. 5 19.
20. 3 21. 22. 23. 24. 5
25.
26. Prepare a dilute starch solution:
a. Pour 0.5 mL from cuvette 1 into cuvette 2.
b. Pour 0.5 mL from cuvette 2 into cuvette 3.
27. Set your spectrophotometer using the control panel:
a. Vis
b. 620 nm
c. A
28. Place cuvette 3 into the loading well of the spectrophotometer. Record the
initial absorbance in your notes.
29. Remove the cuvette from the spectrophotometer. Discard all the cuvettes
into the recycling bin.
Part 2: Preparing the Reaction Solutions
1. Place five clean test tubes onto the workbench.
2. Using items found on the Materials shelf, fill the test tubes according to the
table below. These will include NaH
2
PO
4
(Monosodium Phosphate),
Na
2
HPO
4
(Disodium Hydrogen Phosphate), and NaHCO
3
(Sodium
Bicarbonate):
3. Exp. 2 Reaction Solutions
4. T
e
s
t

T
u
b
e
5. Acet
i
c

A
c
i
d

(
m
L
)
6. NaH2
P
O
4

(
m
L
)
7. Na2H
P
O
4

(
m
L
)
8. W
a
t
e
r

(
m
L
)
9. Na
H
C
O

3

(
m
L
)
10. 2%
Starch
Solution
(mL)
11. 1 12. 6 13. 14. 15. 16. 17. 3
18. 2 19. 20. 6 21. 22. 23. 24. 3
25. 3 26. 27. 5
28. 0
.
5
29. 0
.
5
30. 31. 3
32. 4 33. 34. 3 35. 3 36. 37. 38. 3
39. 5 40. 41. 42. 43. 44. 6 45. 3
46.
47. Attach a pH meter from the Instruments shelf to test tube 1. Record the pH
in your notes.
48. Repeat step 3 with the remaining test tubes, recording the pH of each
solution.
49. Discard the pH meter.
Part 3: Amylase Reactions
First, read the Procedures all the way through.
In this part of the experiment, you'll let an amylase solution digest the 2% starch
solution. Adding this solution to HCl (hydrochloric acid) stops the digestion. You
will then use the spectrophotometer to identify the absorbance of this solution in
iodine.
1. Place two clean cuvettes from the Containers shelf onto the workbench.
2. Fill the first cuvette with 5 mL of HCl and the second cuvette with 5 mL of
Iodine Solution from the Materials shelf. You may choose to label these by
double-clicking on each cuvette.
3. Add 1 mL of Amylase Solution to test tube 1. Immediately click on the clock
icon on the lower left of your workspace to start a timer.
4. After 5 minutes, add 0.5 mL of the reaction solution in test tube 1 into cuvette 1
(containing the HCl). Remember, the HCl will stop the reaction between
the amylase solution and 2% starch solution.
5. Add 0.5 mL of the reaction solution in cuvette 1 into cuvette 2 (containing the
yellow iodine solution).
6. Insert cuvette 2 into the spectrophotometer and record the absorbance in your
lab notes. Be sure that you know the pH of the solution in the original test
tube. You'll need it for experiment 3. Confirm that your
spectrophotometer is set to:
Vis
620 nm
A
7. Repeat the procedure outlined in steps 1 - 6 for each of the five reaction test
tubes. Note: You'll need to prepare two clean cuvettes for each test tube
you use. We suggest you stay organized by staggering the addition of
amylase solution by 3 minutes or more. This strategy will help you to
manage your workbench and to save time.
8. When you are finished, keep the spectrophotometer on the workbench and
discard all other items.
Experiment 3: The Effect of Temperature on Amylase
Enzyme Activity
There is an optimal temperature range for the activity of enzymes. Just as
enzymes are sensitive to pH, they are sensitive to the temperature of their
environment.
You will use the method in Experiment 2 to study the effect of temperature on the
rate of starch digestion by amylase solution. You will still run timed trials, but this
time the temperature will vary while keeping the pH of the reaction solution
constant near 7.2.
1. Place three test tubes from the Containers shelf onto the workbench.
2. To each test tube add the following three components from the Materials shelf:
3 mL NaH
2
PO
4

3 mL Na
2
HPO
4

3 mL 2% Starch Solution
3. Place three constant temperature baths from the Instruments shelf onto the
workbench.
4. Set each temperature bath to a different temperature:
5. Constant Temperature Bath Settings
6. Bath 7. Temperature (C)
8. 1 9. 10
10. 2 11. 40
12. 3 13. 60
14.
15. Move one test tube into each of the baths.
16. Start your timer. After about two minutes the temperature of the solutions
should equilibrate with their baths. You may attach thermometers from
the Instruments shelf to the test tubes to monitor the temperature
change.
17. Place two clean cuvettes from the Containers shelf onto the workbench.
18. Fill the first cuvette with 5 mL of HCl and the second cuvette with 5 mL of
Iodine Solution from the Materials shelf. You may choose to label these by
double-clicking on each cuvette.
19. Add 1 mL of Amylase Solution to test tube 1 in its bath. Immediately click
on the clock icon on the lower left of your workspace to start a timer.
20. After 10 minutes, remove test tube 1 from its bath. Add 0.5 mL of its
reaction solution from test tube 1 into cuvette 1 (containing the HCl) to
stop the reaction.
21. Add 0.5 mL of the reaction solution in cuvette 1 into cuvette 2 (containing
the yellow iodine solution).
22. Insert cuvette 2 into the spectrophotometer. Confirm that your
spectrophotometer is set to:
Vis
620 nm
A
23. Record the absorbance, bath temperature, and pH (7.2) in your lab
notes.
24. Discard the two used cuvettes.
25. Repeat steps 7 - 14 for the remaining test tubes. Remember to save your
notes.

Something Practical: Why Does it Sting When You
Eat Pineapple?
Pineapple contains Bromelain, a mixture of protein-digesting enzymes. When
you eat a citrus fruit like a pineapple, the enzymes start to digest the proteins in
your tongue and mouth. That's why it stings. Don't worry, there aren't enough
enzymes in pineapple to damage your mouth, just enough to make it sting.
Bromelain is used as a meat tenderizer because the enzymes break down the
proteins in the meat, making it softer. But you should be careful when using
Bromelain, or mixing pineapples with meat: If you leave it too long the enzymes
will break down so many of the proteins in the meat that it will become mushy
and unappealing instead of pleasantly tender. If you're a fan of T.V. cooking
shows like "Top Chef", you've probably seen the judges yelling at one of the
contestants for mixing pineapple into a ceviche and serving them mushy fish.