CHEM 137.

2 – BIOCHEMISTRY I (LABORATORY)
LABORATORY REPORT

Name: Reynand E. Corpuz Date Submitted: 02-07-2021

Lab Schedule: TTh 8:00 – 10:00 Date Performed: ____________

Exercise No. 7
Enzyme Activity
I. OBJECTIVES:
At the end of the experiment, the students should be able to associate the presence of enzymes
with the catalysis of chemical reactions living in cells; and to determine the effect of enzyme
concentration, substrate concentration, pH and heavy metal upon the activity oof salivary amylase.

II. RESULTS AND DISCUSSION:

A. Enzyme Concentration

Enzyme Activity
Concentration Time for Starch Hydrolysis (s)
Rate of reaction (1/s)

0.2 58 0.01724

0.4 40 0.025

0.8 40 0.025

Graph of Enzyme Activity vs. Enzyme Concentration

Enzyme Activity

0.03

0.025

0.02

0.015

0.01

0.005

0
0.2 0.4 0.8
 Enzymes are molecules that speed up the rate of specific biochemical reactions in our cell
by lowering the activation energy needed for the reaction to occur. The concentration of enzyme
affects the rate of reaction directly, that means increasing the concentration of the enzymes would
mean an increase in the reaction rate because the probability of the enzyme to bind with substrate
forming Enzyme-Substrate complex is higher; this is true for any catalyst in which enzymes are
classified.
In the experiment, three (3) solutions containing different concentration of pancreatic
amylase enzyme were prepared for investigation. The samples were prepared through serial
dilution method where in the succeeding amylase solutions contain one-half concentration of
amylase enzyme of the preceding solutions. The sample solutions were then subjected to iodine
test in which the appearance of a deep-blue solution indicates a positive result for the test which
confirms the presence of starch. In the test, the amylase solutions were added with 1 mL starch
and upon addition, timer was started and a spot of the amylase + starch solution were added in the
spot of the iodine solutions (every 10 seconds) in the spot plate until the solution no longer changes
color. Based on the result (see table above), it was observed that the solution with the concentration
of 0.2 takes about 58 seconds for the starch to be completely digested by the enzyme. It is true
because 0.2 is the lowest concentration so the activity of the enzyme is expected to be the slowest.
However, the time for the 0.4 and 0.8 amylase solutions to completely digest the starch were
essentially the same at 40 seconds. The enzyme activity does speed-up and is an improvement
from the result of 0.2, but in theory, the 0.8 concentration would have digested the starch faster
than the 0.4 because its concentration is a half higher. Since that is the result, then the only
explanation is that the substrate which is the starch must have all been bounded by the amylase at
concentration of 0.4 therefore, further addition would not change anymore since all starch
molecule is already used up. Since the substrate were all used up, the reaction rate will terminate
as it is dependent with the time.

B. Effect of pH

Enzyme Activity
pH Time for Starch Hydrolysis (s)
Rate of reaction (1/s)

4 390 0.00256

7 90 0.0111

9 660 0.00152
 The pH of the solution can also affect the enzyme activity. In our body, enzyme works in
a narrow optimum pH range thus, enzyme activity will be affected beyond this range. Since most
enzymes are proteins, enzymes are expected to be sensitive to pH change because the amino acid
component of the enzyme, specifically the positively and negatively charged groups will attract
which contributes to the enzyme folding that change the shape of its active site and will slows the
enzyme activity. Similarly, extreme pH can denaturize the enzyme and so, will have a great effect
on its activity.

0.012

0.01

0.008

0.006

0.004

0.002

0
4 7 9

Enzyme Activity

Graph of Enzyme Activity (Rate of reaction) vs. pH

In this part of the experiment, three (3) amylase + starch solutions at pH 4, 7 and 9 were
prepared to examine the effect of pH to the enzyme activity. The same with the previous
experiment, the sample solutions were also subjected to iodine test where in a spot of the amylase
+ starch solution were added in the spot of the iodine solutions (every 30 seconds) in the spot plate
until the solution no longer changes color. Based on the results, it was observed that enzyme
activity was highest at pH 7, and the enzyme activity relatively drops at pH 4 and 9. Since, the
optimum working pH range of amylase is at pH 6.7-7.0, then enzyme activity at pH 7 would not
be affected by the condition as it is the best state where the amylase can perform with excellent
outcome. However, beyond this range such as pH 4 and 9, enzyme activity will be affected
negatively. Henceforth, the results are plausible and accurate.
C. Effect of Temperature
Enzyme activity
Temperature Time for Hydrolysis (s)
Rate of reaction (1/s)

The blue - black colour did not

0°C 0
disappear

20°C 360 0.00278

40°C 240 0.00417

60°C 120 0.00833

The blue – black colour did not

80°C 0
disappear

The blue – black colour did not

100°C 0
disappear

Enzyme Activity

0.009
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0
0 20 40 60 80 100

Graph of Enzyme Activity (Rate of reaction) vs. Temperature

In this part of the experiment, the effect of temperature to the enzyme activity was analyzed
by placing the amylase and starch solutions to five beakers with temperature of 0 oC, 20oC, 40oC,
60oC, 80oC, and 100oC before reacting with each other. The samples underwent iodine test, same
with the previous two, where in a spot of the amylase + starch solutions were added in the spot of
the iodine solutions with interval of 2 minutes, in the spot plate until the solution no longer changes
color.
For most chemical reactions, the increase in temperature will result to an increase in
reaction rate. Enzymes somehow follow this principle, however, further increase of temperature
 to extreme level (approximately above 50oC) can denatures the protein and disturb the active site
causing the reaction rate to slow down. This is the concept behind the observed result (see graph)
because as the temperature was increased, the enzyme activity also increased but above 60 oC, the
enzyme activity suddenly falls to 0. At this moment (80oC and 100oC), all of the enzymes are being
denatured therefore, gives negative result in the test.

D. Inhibition of Enzyme Activity

Color Change
Test tube HgCl2 (Present/Absent)
Initial Color Final Color

A Present Yellow Yellow

B Absent Yellow blue

The effect of inhibitor to the enzyme activity was investigated in this part of experiment.
In this part of experiment, two (2) solutions were prepared; test tube A is a solution of urea,
bromothymol indicator, mercuric chloride, and urease; while test tube B is a solution of urea,
bromothymol indicator, distilled water, and urease. After 30 minutes, it was observed that the
solution B changed color from yellow to blue which indicates positive reaction. The urea in this
solution has been successfully digested by the urease enzyme that is why the solution turned blue.
On the other hand, the solution B remained yellow even after the span of 30 minutes and it is due
to the inhibitor HgCl2 which affects the enzyme activity. Inhibitors affect the enzyme activity
because as it binds to the enzyme’s active sites, it reduces the compatibility of the enzyme to bind
with the substrate to form Substrate-Enzyme complex, therefore decreasing the reaction rate.
Mercury is an active inhibitor that inhibits enzyme activity by binding to sulfhydryl groups found
in urease, specifically on the amino acid cysteine, one of its constituent amino acid. The inhibition
of mercury decreases the reaction rate (sometimes to zero) of urease and urea and it is evidently
accurate with the experiment.

III. ANSWERS TO QUESTIONS

1. (A) How does the activity of the enzyme change as its concentration is increased? What are some
reasons for these changes in activity?
Answer:
If the concentration of the enzyme is increased, the enzyme activity is also increased. The
reason for the change in activity is that enzyme affects the rate of reaction directly, that means
 increasing the concentration of the enzymes would mean an increase in the reaction rate because
the probability of the enzyme to bind with substrate forming Enzyme-Substrate complex is higher;
this is true for any catalyst in which enzymes are classified.

2. (B) What is the effect of pH upon the relative enzyme activity? What are some reasons for this
effect?
Answer:
Enzyme can work properly in a narrow pH range but beyond this range, enzyme activity
slows down. Since most enzymes are proteins, enzymes are expected to be sensitive to pH change
because the amino acid component of the enzyme, specifically the positively and negatively
charged groups will attract which contributes to the enzyme folding that change the shape of its
active site and will slows the enzyme activity. Similarly, extreme pH can denaturize the enzyme
and so, will have a great effect on its activity.

3. (C) How is the enzyme affected by low temperatures? By high temperatures? What are some
reasons for these temperature effects? According to your experiment, what is the optimum
temperature for salivary amylase?
Answer:
In low temperature, enzyme activity is slow. At high temperature, enzyme activity is also
increased, however, further increase in temperature slows down the enzyme activity. The reason
for this is that at very high temperature, enzyme is denatured causing the shape of the enzyme to
be altered. Based on the experiment, the optimum temperature for salivary amylase is pH 7.

4. (D) Which of the compounds added to the reaction tubes are inhibitors? Why do these compounds
act as inhibitors of enzyme activity? Why is an alcohol swab used prior to giving an injection to a
patient?
Answer:
The HgCl2 is the inhibitor among the added compound in the reaction tubes. This
compound act as inhibitor of enzyme activity because it binds on the enzyme’s active site and
reduces the compatibility of the enzyme to bind with the substrate to form Substrate-Enzyme
complex, therefore decreasing the reaction rate. Alcohol is used to disinfect the skin prior to
injections to prevent infections caused by bacteria on the skin being injected within tissue.
IV. CONCLUSIONS:
The focus of the experiment was to associate the presence of enzymes with the catalysis of
chemical reactions in living cells; and to determine the effect of enzyme concentration, pH,
temperature, and heavy metal upon the activity of amylase. With the results, it is generalized that in
our body, enzymes can speed up the rate of biochemical reactions but at the conditions were the
enzyme concentration is low, extreme pH, high temperature, and presence of inhibitors could decrease
the activity of the enzyme. In the experiment, it was found out that enzyme concentration has a direct
effect on enzyme activity. The pH of the enzyme solution also affects the enzyme activity in a way
that adjusting the pH beyond the working range of amylase would decrease the enzyme activity.
Moreover, it was observed that temperature increases the enzyme activity of the amylase, however,
further increase to extreme level would make the enzyme activity to slow down. Lastly, enzyme
inhibitor such as mercury obstructs the enzyme to pair with the substrate therefore, also decreasing the
enzyme activity substantially.