Experiment 12

FACTORS THAT INFLUENCE THE ACTIVITY OF AN ENZYME

Michael E. Pugh and Wayne P. Anderson
(Rev. 3/2004)

*Adapted from Experiment 74, “Factors Affecting Enzymatic Activity” in John R. Holum and Sandra L.
th
Olmstead, Laboratory Manual for Fundamentals of General, Organic and Biological Chemistry, 5 Ed.,
New York: Wiley, 1994

INTRODUCTION

Amylase, an enzyme that is found in saliva, catalyzes the hydrolysis of starch (amylase). Since enzymes
are proteins, their secondary and tertiary structures are affected by temperature, pH, and the presence of
heavy metal ions. Enzyme activity is closely associated with the structure of an enzyme, so any change
in the secondary or tertiary structure leads to a change in enzyme activity. In this experiment you will
examine the effect of temperature and pH on the activity of amylase. Molecular iodine forms a complex
with starch that has a characteristic deep blue color. As starch undergoes hydrolysis to form
oligosaccharides and glucose, the characteristic of color of the starch-iodine complex disappears.
Therefore, loss of the deep blue color can be used to measure of the extent of hydrolysis of starch. A
second test for hydrolysis is the occurrence of a hydrolysis is.

PART I. EFFECT OF TEMPERATURE ON ENYZME ACTIVITY

In order to make sure that the concentrations of enzyme and starch remain reasonably constant in
different parts of the experiment, use an eye dropper to measure quantities of solutions. Assume that 20
drops represent 1.0 mL. Measure and record the room temperature.

1. a. Label three medium test tubes as “0”, “rt” and “100”. Into each test tube place
2.5 mL of buffered starch solution and 2.5 mL of distilled water. Place the test
tube marked “0” into the ice bath, the one marked “rt” leave at room temperature, and the one
marked “100” into a beaker of boiling water.

b. Place 1 mL of freshly prepared amylase solution (100 mg/100 mL) into each of
three small test tubes. Put one of these into the ice bath, another left at room temperature, the
third into the beaker of boiling water.

c. Allow the solutions to remain in the temperature baths for about 5 minutes to equilibrate.
o
2. Carry out the following steps in sequence for the solutions in the 0 C, rt and 100° temperature
baths.

a. Remove the test tube containing the starch and the test tube containing the enzyme from the bath
and pour the enzyme solution into the starch solution. Record the time to the nearest second,
and call this starting time 0. Quickly place a piece of parafilm over the end of the test tube, invert
mix thoroughly, remove the parafilm and remove a small amount of the solution from the test tube
using a disposable transfer pipette. Place the remaining solution back into the temperature bath.
Place 4 drops of the solution onto a spot place that contains 1 drop of the iodine solution. Record
the color. Put any excess starch/enzyme solution in the pipette back into the test tube in the
temperature bath; leave the transfer pipette in the solutions.

b. Take a sample from the solution using the disposable pipette exactly 1 minute following time 0.
Place 4 drops onto a second spot of the spot plate containing 1 drop of iodine solution. Record
the color. Return any excess starch/enzyme solution in the pipette to the test tube in the
temperature bath; leave the transfer pipette in the solutions.

c. Repeat the procedure in part (2b) at the following time intervals until the color of the solution
following addition of the iodine is yellow: 2 min, 4 min, 6 min, 8 min, 10 min. If the color of the
 solution in the spot plate remains yellow for successive trials, it is not necessary to continue that
particular reaction.

PART II. EFFECT OF pH ON ENZYME ACTIVITY

1. a. Label three medium test tubes as “5”, “7”, and “9”. Into the test tube marked “5”, place 2.5 mL of
unbuffered starch solution and 2.5 mL of pH 5 buffer solution. Into the test tube marked “7”, place
2.5 mL of unbuffered starch solution and 2.5 mL of pH 7 buffer solution. Into the test tube marked
“9”, place 2.5 mL of unbuffered starch solution and 2.5 mL of pH 9 buffer solution. Leave all three
of these substrate tubes at room temperature.

b. Place 1 mL of the amylase solution into each of three small test tubes.

c. Allow the solutions to remain at room temperature bath for 5 minutes to equilibrate.

2. Carry out the steps 2a-2c in PART I in sequence for the solutions that are buffered at pH 5, 7, and 9.

PART III. EFFECT OF METAL IONS ON ENZYME ACTIVITY

2+ 3+ 2+ 2+ 2+ +
In this part you will test the effect of a metal ion on enzyme activity. Cu , Fe , Zn , Pb , Hg , Ag .
Your instructor will assign your group one of these.

1. a. Label a test tube with the identity of the metal ion in the salt solution you will use for this part. Into
the test tube, place 2.5 mL of buffered starch solution and 2.5 mL of the metal ion solution.
Leave your substrate/metal ion solution at room temperature.

b. Place 1 mL of the amylase solution into a small test tube.

c. Allow the solutions to remain at room temperature bath for about 5 minutes to equilibrate.

2. Carry out the steps 2a-2c in PART I in sequence for the solutions containing the metal ion.

3. Compare your metal ion results with the results of the other metal ions tested.
 52-105
Enzyme Activity

Name ________________

Record the colors of the starch/enzyme solution in the presence of iodine.

PART I.

Time (Minutes) 0° Room Temperature 100°

T= _______
0
1
2
4
6
8
10

PART II.

Temperature ____________

Time (Minutes) pH=5 pH=7 pH=9

0
1
2
4
6
8
10

PART III.

Temperature _________

Time (minutes) Metal Ion = _________

0
1
2
4
6
8
10

INTERPRETATION

1. Give a clear explanation of the effect of temperature on the activity of amylase.

Consult your class notes for ideas.

2. Give a clear explanation of the effect of pH on the activity of amylase. Consult your
class notes for ideas.

3. Give a clear explanation of the effect of the metal ion on the activity of amylase. Be
sure to compare the results of all of the metal ions.