Effects of Change in pH and Temperature on the Reaction Rates of Enzyme-Catalyzed Reaction

Marcus Natividad, Barbara Ngo, Lexley Ong and Jane Jenelle Quilaneta
Group 8 2C Pharmacy Biochemistry Laboratory

ABSTRACT
In this experiment, the glucose assay using a DNS colorimetric method measured and showed the maximum capacity
of invertase activity in increasing concentration of the standard through a graphical representation, the best fit straight line. Factors that
affect the invertase is also investigated such as pH and temperature .The results obtained were represented by bell-
shaped graphs that shows the optimum amount of pH and temperature.

INTRODUCTION
This experiment aims to make the students
able to determine the effects of change in pH and
temperature on the reaction rates of an enzyme-
catalyzed reaction.

Living organisms are composed of intricately
systems of chemical reaction. Most of these
chemical reactions are catalyzed by enzymes that
allow these reactions to proceed at a rate
sufficient to sustain life. Enzyme accelerates
reactions without being changed themselves [1].
It is protein molecule that is a biological
catalyst with three characteristics. First, which is
its basic function is to increase the rate of a
reaction. Second, most enzymes act specifically
with only one reactant called a substrate to
produce products. The third and most remarkable
characteristic is that enzymes are regulated from
a state of low activity to high activity and vice
versa [2].
Sucrose, commonly known as table sugar, is a
disaccharide composed of an alpha-D-glucose
molecule and a beta-D-fructose molecule linked
by an alpha-1,4-glycosidic bond. When this bond
is cleaved in a hydrolysis reaction, an equimolar
mixture of glucose and fructose is generated.
This mixture of monosaccharide is called invert
sugar, which is derived from the fact that sucrose
rotates plane polarized light to the right i.e.,
dextrorotatory, +66.5, whereas the hydrolysis
products rotates plane polarized light to the left
i.e., levorotatory, -20 .
Sucrose can be hydrolyzed in the presence of
an enzyme called invertase or sucrose which
shown in the equation:
Sucrose + H
2
O ---> glucose + fructose
The official name for invertase is beta-
fructofuranosidase, which implies that the
reaction catalyzed by this enzyme is the
hydrolysis of the terminal non-reducing beta-
fructofuranoside residues in beta-
fructofuranosides[3].

Invertase is usually found on plants. It acts as
a catalyst for the hydrolysis of sucrose. It is a
significant enzyme because glucose is an
important product of photosynthesis. Invertase is
also used in the confectionery industry where
fructose is preferred over sucrose because it is
sweeter and does not crystallize easily[4].
EXPERIMENTAL
A. Sample Used
Sample Used: Glucose, Dinitrosalicyclic reagent

B. Procedures
1. Sucrose Assay Using Dinitrosalicyclic
Method

First, We prepared series of test tubes.All
these test tubes were covered with marbles to
prevent evaporation of solvent.Then, we added 3
drops (approximately 0.05ml) concentrated HCl
to each tube and mixed it. It was incubated at
90C water bath for 5 minutes. Then an addition
of 0.15 mL of KOH was done to neutralize the
solution. Next, is the addition of 1.5 mL of 0.1 M
buffer solution, pH 5 and was mixed.Then , 3 mL
of DNS reagent was added. The test tubes were
immersed in a 95C water bath for 10 minutes to
develop the characteristic red-brown color. After
the solution has been cooled, the absorbance at
540nm was measured. Finally, the hydrolized-
sucrose standard curve was constructed by
plotting A
540
against concentration (mg/mL).

2. Effect of pH on Invertase Activity

First, we prepared 6 numbered test tubes and a
blank test tube. Then, we added 1.4 mL
appropriate 0.1 M buffer solution to each test
tube. For the blank test tube we just added 3mL
of distilled water, for test tube number 1 we
added pH 1.00, for test tube number 2 we added
pH 3.00, for test tube number 3 we added pH
5.00, for test tube number 4 we added pH 7.00,
for test tube number 5 we added pH 9.00 and for
test tube number 6 we added pH 11.00. After
adding the desired amount of pH to each test
tube, 0.10 mL enzyme stock solution was added
to each test tube except for the blank test tube.
It was mixed thoroughly and was incubated in
60C water bath for 5 minutes. Next, is the
addition 1.50 mL of sucrose solution and was
incubated reaction mixture in 60C water bath for
5 minutes. Then, 3 mL of Dinitrosalicyclic reagent
was added. The test tubes were immersed in 5C
water bath for 10 minutes to develop the
characteristic red-brown color. Then the
absorbance at 540 nm was measured. Finally, the
amount of sucrose hydrolyzed was determined
using hydrolyzed-sucrose standard curve that
was constructed in the Dinitrosalicyclic
colorimetric method.

3. Effect of Temperature on Invertase
Activity
To test the effect of temperature on Invertase
activity, our group placed 20, 30, 50, 60, 70 and
90C water baths and prepared 6 test tubes with
each test tube containing 1.5 mL sucrose
solution. The test tubes were incubated
separately for 5 minutes in each water bath. In
another test tube, 0.80 mL enzyme stock solution
was mixed with 19.20 mL 0.1M buffer solution,pH
5. Then, we added 3 mL of diluted enzyme
solution to all tubes. It was incubated for another
5 minutes. Remember not to remove the test
tubes from their respective water baths. Then, 3
mL of DNS reagent was added. The test tubes
were immersed in a 95C water bath for 10
minutes to develop characteristic re-brown color
and then it was cooled down. Remember that all
the test tubes are covered with marble to prevent
evaporation of solvent. Meanwhile, another
member prepared a bank solution by repeating
all the steps but instead of adding enzyme stock
solution, denatured enzyme is used. Then, the
absorbance at 540nm is measured. The amount
of sucrose hydrolyzed was determined by using
sucrose standard curve constructed in the
dinitrosalicylic colometric method.
RESULTS AND DISCUSSION

Glucose, and other reducing sugars,reacts with DNS by
oxidation reaction producing a colored compound that
shows a maximal molar extinction at 530nm.Glucose is
reduced into gluconic acid,same as to dinitrosalicylic
acid to 3-amino,5-nitro saliccycli acid,giving the
characterisctic red-brown color. Oxidation is a reversible
chemical reaction in which one of the reactions is
oxidation and the reverse reaction is reduction. Since
the absorbance at 540 nm is linearly dependent on the
concentration or mass of glucose the reaction can be
used for quantification of reducing sugar

In table 1, We were able to measure the amount of light
transmitted through a sample at a given wavelength.
The absorbance of blank test tube was subtracted to the
absorbance of the numbered test tubes. This was made
to measure only the absorbance of the invertase,
without the additional reagents.
Test tube no Amount of Acid
hydrolyzed
glucose
(mg/ml)
Absorbance
blank 0 2.35
1 0.5 2.10-2.35
=-0.25
2 0.83 2.96-2.35
=0.61
3 1.33 3.04-2.35
=0.69
4 1.67 3.07-2.35
=0.72
5 2.17 3.10-2.35
=0.78
6 2.50 3.11-2.35
=0.76
Table 1.Glucose Assay Using Dinitrosalicyclic
Method

The graph below is the glucose calibration
curve. It was shows the presence of glucose in
the solution and the rate of formation of glucose.
It is illustrated that the absorbance is directly
proportioned to the amount of hydrolyzed
glucose which means that the higher the
absorbance the higher amount of acid hydrolyzed
glucose is present.


Figure 2. Linear Graph of Glucose Assay Using
Dinitrosalicylic Method
The Invertase can be affected by the changes in
pH. An Extremely high or low pH values generally
result in complete loss of activity for most
enzymes.


0
0.2
0.4
0.6
0.8
1
0 1 2 3
A
b
s
o
r
b
a
n
c
e
Glucose mg/ ml
Glucose Assay
pH Amount of
Acid
hydrolyzed
sucrose
(mg/ml)
Absorbance
blank 0.223 A
3 0.5 1.652-0.233
=1.419 A
4 0.34 1.652-0.233
=0.032 A
5 0.28 1.580-0.233
=1.347 A
7 0.36 1.660-2.33
=1.427 A
8 0.26 1.601-0.233
=1.368
11 0.02 1.459-0.233
=1.226
Table 2. Effect of pH on Invertase Activity
In figure 3, A bell shaped graph was formed. The
most favorable pH value, which is the point
where the enzyme is most active, is known as the
optimum pH is at pH 6. It shows that at pH 7 and
above it the concentration of invertase will slope down
indicating denaturation of enzyme. It indicates that
invertase reactivity is controlled and occurs to alimited
extent.

Figure 3. Graph of the effect of pH on Invertase
Activity
The temperature affects the invertase activity.
Too cool will not allow the enzyme to react,
Too hot will denature the protein.

Temperature Amount of
Acid
hydrolyzed
sucrose
(mg/ml)
Absorbance
20 0.05 1.339-0.213
=1.126
30 0.1 1.459-0.213
=1.246
50 0.12 1.52-0.213
=1.367
60 0.32 2.88-0.213
=2.667
70 0.18 1.799-0.213
=1.586
90 0.17 1.762-0.213
=1.549
Table 3. Effect of Temperature on Invertase
activity
Optimum Temperature was obtained at
60C . It infers that the activation energy is spontaneous
that the transition state was inferred to the highest value
of absorbance in the graph. The highest peak of the curve
represents the highest concentration which was
determined by the Absorbance. Temperature above 60C
shows that the invertase reached denaturation.


Figure 4. Graph of Temperature on Invertase
Activity

REFERENCES
[1]Crisostomo A. et.al.(2010).Laboratory Manual
in General Biochemistry.Quezon City: C&E
Publishing
Inc.do.edu/hndbksupport/ochemlabtech.html
2003
[2]http://www.elmhurst.edu/~chm/vchembook/5
70enzymes.html
[3]http://www.eng.umd.edu/~nsw/ench485/lab1
4.htm
[4]Campbell N.A.;Reece
J.B..(2002).Biology,6
th
edition.San Francisco,USA:
Benjamin Cummings.



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