Determination on the Effect of Temperature on Invertase Activity Using

Spectrophotometry

By: Prince Dario De Guzman
5
, Margarita Jai B. Cobangco
1
Ernesto Rafael C. Dayrit
2
, Denise Victoria DG. De
Guzman
3
, and Mellanie Claire C. De Guzman
4


Group 3, 2F Medical Technology, Faculty of Pharmacy, University of Santo Tomas, Espaa, Manila


Abstract

This experiment seeks to analyze, evaluate, and validate the effects of temperature on invertase activity.
Enzymes are proteins that act as catalysts by lowering the activation energy in specific chemical reactions. A specific type
of enzyme, Invertase, hydrolyses sucrose into glucose and fructose. To determine the effects of temperature, two different
kinds of enzymes denatured and stock solution were both immersed in varied temperatures of hot water bath to facilitate
hydrolysis of sucrose. Dinitrosalicylic acid works on reduction-oxidation method and due to its change in coloration;
absorbance can be measured using spectrophotometric analysis. The absorbance was measured by setting the
wavelength of the standard samples at 540 nm and zero for its corresponding blank solutions. The data gathered showed
the relationship of absorbance to the temeperature and acid-hydrolyzed sucrose concentration. Consequently, it was
inferred that enzymes have an optimal temperature at which they function most effectively.


Introduction
Living cells is the site of tremendous
biological activity called metabolism. This is the
process of chemical and physical change
which goes on continually in the living
organism. Tissue repair, conversion of food to
energy, excretion of wastes material and
reproduction are the activities that are
associated with life and majority of these
activities are not spontaneous.
Catalysis makes these possible which is
necessary in all life forms. It is the acceleration
of a chemical reaction through the presence of
some substances that in which itself undergoes
no permanent chemical change. The catalysts
of biological reactions are enzymes.
An enzyme is a biomolecule, usually a
protein, that acts as a biological catalyst to
speed the rate of a biochemical reaction
(Boyer, 2006) which has the following
characteristics: First, the basic function of an
enzyme is to increase the rate of a reaction.
Second, most enzymes act specifically with
only one reactant (called a substrate) to
produce products. Third, enzymes are
regulated from a state of low activity to high
activity and vice versa. [2] Enzymes are
affected by changes in pH (Campbell & Reece,
2002). Extreme pH values result in loss of
activity for most enzymes. Furthermore, there is
a most favourable pH for enzyme the point
where enzyme is most active. This point is
known as the optimal pH.
There are different classification for
enzymes: Oxidases or Dehydrogenases for
Oxidation-reduction reactions, Transferases
for Transfer of functional groups, Hydrolases
for Hydrolysis reactions, Lyases for Addition to
double bonds or its reverse, Isomerases for
Isomerization reactions, and Ligases or
Synthetases Formation of bonds with ATP
cleavage
Invertase, or beta-fructofuranosidase, is
an enzyme derived from yeast. It catalyzes the
hydrolysis (breakdown) of sucrose, which is a
table sugar, down into the simple sugars
glucose and fructose. The resulting product,
also known as inverted sugar syrup. Invertases
cleave the O-C(fructose) bond. Though the
enzyme is inclined to higher levels of activity at
low temperatures, it is best when used at about
60
o
C, because sucrose will split faster at higher
temperatures. [3]
Baker's yeast is the common name for
the strains of yeast commonly used as a
leavening agent in baking bread and bakery
products, where it converts the fermentable
sugars present in the dough into carbon
dioxide and ethanol. Baker's yeast is almost
always of the species Saccharomyces
cerevisiae,
3,5-Dinitrosalicylic acid (DNS or DNSA,
IUPAC name 2-hydroxy-3,5-dinitrobenzoic
acid) is an aromatic compound that reacts with
reducing sugars and other reducing molecules.
It was first introduced as a method to detect
reducing substances in urine and has since
been widely used, for example, for
quantification of carbohydrates levels in blood.
It is mainly used in assay of alpha-amylase.
However, enzymatic methods are usually
preferred to DNS due to their specificity.
Disaccharides are compounds that
contain a bond between carbon of one sugar
and a hydroxyl group at any position on the
other sugar. Sucrose is the only non-
reducing common disaccharide. Consequently,
test for sugar detection using DNS (3,5-
dinitrosalicylic acid) solution result in negative
readings for sucrose. However, this method
can still be applied if sucrose is first hydrolyzed
in an acid solution to yield glucose and
fructose.
This experiment seeks to evaluate and
validate the optimum temperature of an
invertase activity and analyze the possible
outcomes by controlling different variables with
the use sensitive technique like
spectrophotometry and linear regression
analysis.
Materials and Methods
Series of water baths were prepared
with varied temperatures; 20, 30, 50, 60, 70,
and 90C. Then, two sets of six test tubes were
prepared. For the first set which served as
standard, 1.5 mL of sucrose solution was
added followed by separate incubation in each
water bath for 5 minutes. While incubating, a
diluted enzyme stock solution was prepared in
a separate test tube by mixing 0.80 mL
enzyme stock solution with 19.20 mL 0.1 M
buffer solution at pH 5. After dilution, 3 mL of
the diluted enzyme was added to each test tube
followed by incubation for 5 minutes. After
incubating, 3 mL of DNS 9 3,5-dinitrosalicylic
acid) reagent was added to the test tubes,
followed by immersion of test tubes in 95C
water bath for 10 minutes in order to develop
the red brown color characteristic.













Figure 1. Red-brown coloration after the water bath.

The same procedure was done in the
second set of test tubes; the only difference is
that denatured enzyme was added instead of
enzyme stock solution.

Table 1-1. Components of Standard Set of Test Tubes
Test
tube
Temperature
(C)
Enzyme stock
solution
Denatured
enzyme
1 20 + -
2 30 + -
3 50 + -
4 60 + -
5 70 + -
6 90 + -

Table 1-2. Components of Blank Set of Test Tubes
Test
tube
Temperature
(C)
Enzyme stock
solution
Denatured
enzyme
1 20 - +
2 30 - +
3 50 - +
4 60 - +
5 70 - +
6 90 - +

After the cooling process, all solutions
were transferred to separate cuvettes. The
absorbance was measured by using a
spectrophotometer with 540 nm wavelength.
The absorbance of the blank solution was put
to zero. The resulting absorbances were
tabulated and the concentration of each
hydrolyzed sucrose was determined using the
standard curve constructed in the
dinitrosalicylic colometric method. All the data
collected were tabulated and interpreted using
figures and tables.

Results and Discussion
A stock solution is a concentrated
solution to be diluted to some lower
concentration, whereas, denatured enzyme
have distorted active sites therefore disabling
the enzyme to lock up with a substrate.
Denaturing an enzyme was done by subjecting
it to high temperature where they vibrate
vigorously that the bonds holding the protein
structure in its specific shape becomes broken.








Figure 2. Hydrolysis of Sucrose into Glucose and
Fructose by invertase.

Sucrose is made up of glucose and
fructose and is connected by glycosidic
linkage. Upon hydrolysis the glycosidic bond is
broken and results in formation of glucose and
fructose. The hydrolysis reaction takes place at
slower rate, but upon addition of enzyme
invertase the reaction takes place at rapid rate.
Dinitrosalicylic acid reacts with reducing
sugars and other reducing moleculesto form 3-
amino-5-nitrosalicylic acid, which absorbs light
strongly at 540 nm thus the amount of enzyme
in the reaction mixture therefore had to be
rather large in order produce sufficient reducing
sugar for color development that would still be
detectable. This method tests for the presence
of free carbonyl group (C=O), the so-called
reducing sugars. This involves the oxidation of
the aldehyde functional group present in, for
example, glucose and the ketone functional
group in fructose. Simultaneously, 3,5-
dinitrosalicylic acid (DNS) is reduced to 3-
amino,5-nitrosalicylic acid under alkaline
conditions.
One of the factors in the stability of
enzymes is temperature. The optimum
temperature of the invertase activity should be
at exactly 60C (140F). If the invertase was
exposed in elevated temperature and extreme
pH after reaching its optimum, the enzyme will
slope down indicating denaturation of enzyme.
This phenomenon indicates that enzymes
reactivity is controlled and occurs to a limited
extent in the biological reactions.

Table 2-1. Absorbance obtained
Temperature
(C)
Absorbance
540
20 0.33
30 0.216
50 -.026
60 0.678
70 0.410
90 -.124


Figure 3-1. Graph of Absorbance vs. Temperature

-0.2
0
0.2
0.4
0.6
0.8
0 20 40 60 80 100
A
b
s
o
r
b
a
n
c
e
,

5
4
0

n
m
Temperature (C)
Effect of Temperature on Invertase Activity
Based on the data presented, the group
got a negative value of absorbance in test tube
3 and 6 with temperatures of 50 and 90C.
However, a bell-shaped curve was still
obtained. Theoretically speaking, the linear
sequence must continue on raising until it
reaches the peak or the optimum temperature.
As the temperature continue to increase
surpassing the optimum value which is 60C,
the linear sequence will start to drop indicating
the start of denaturation of the enzyme.
There are many possibilities of having a
negative absorbance; one is that the samples
might have been contaminated due to improper
cleaning of laboratory apparatus such as test
tubes, cuvettes, and pipettes. The Dilution
process might not have been successfully
done because of human error in measuring the
specific volume. Also, the group had a limited
time in performing the experiment thus the
needed temperature for each water bath has
not been successfully provided.
The concentration of the hydrolyzed
sucrose under different temperatures has been
determined by the analysis in the result of the
Sucrose assay using dinitrosalicylic colometric
method from the other group.

Table 2-2. Conc. And Absorbance of HSC from DNS
Colorimetric Method
Test
Tub
e
Amount of
Hydrolyzed Sucrose
(mg/mL)
Absorbance
540
Blan
k
0 0
1 1.67 -0.013
2 3.33 -0.007
3 5 0.001
4 6.67 0.009
5 8.33 0.347
6 10 0.816



Figure 3-2. Graph of HSC in DNS Colorimetric Method
note: The calibration curve is (y=0.043x)

The negative value of absorbance is also
due to human errors in handling the apparatus
and reagents. The conc. (C
2
) In the table is
derived from the dilution formula wherein
C
1
=10mg/mL, V
1
= (specific volume of sucrose
standard solution), and V
2
= total volume which
is 1.5 mL.
By assigning the x-value to the derived
conc. and the y-value to the absorbance, a
graph with a linear sequence has been made.
With the use of the calibration curve of that
graph, the group was able to derived the
specific concentration of the hydrolyzed sucrose
under different temperatures.

Table 2-3. Derived Concentration from
calibration curve

By substituting the specific absorbance
to the y-value of the calibration curve divided by
the value of 0.043, the group has derived the
specific concentration which is x.


y = 0.0438x
R = 0.4944
y = 0.0438x
R = 0.4944
-0.5
0
0.5
1
0 5 10 15
A
b
s
o
r
b
a
n
c
e
,

5
4
0

n
m
Concentration, mg/mL
Absorbance- Hydrolyzed Sucrose
Concentration
Test
Tube
Temperature
(C)
HSC
(mg/mL)
Absorbance
540
nm
1 20 7.67 0.33
2 30 5.02 0.216
3 50 -0.604 -.026
4 60 15.8 0.678
5 70 9.53 0.410
6 90 -2.88 -.124


y which is the speciic absoibance

Figure 3-3. Graph of Derived Conc. vs. Temperature

This figure above proved that the
derived concentration from the calibration
curve is correct due to the bell-like orientation
formed by the linear sequence of the graph.

References
From books:

1. Atkins, Robert C. & Francis A.Carey. (2002). Organic
Chemistry: A Brief Course (3rd ed.). Mcgraw-Hill
Companies,inc. USA,NY pp310,313

2. Boyer, Rodney. (2006). Concepts in Biochemistry (3
rd
ed.)
John Wiley and Sons Asia Pte Ltd. 131- 155

3. Campbell, M.K. & Farell, S.O. (2009). Biochemistry (6
th
ed.)
Philippines: Cengage Learning Asia Pte Ltd. 143- 150

4. Mckee, J.R. & Mckee, T. (2009). Biochemistry: The
Molecular Basis of Life (4
th
ed.). New York: Oxford University
Press. 184- 195, 202- 219

5. Reiner, J.M. (1969). Behavior of enzyme Systems. New
York: Van Nostrand Reinhold. 45- 54, 113- 116, 261- 284.

From online websites and researches:

6. http://www.eng.umd.edu/~nsw/ench485/lab14.htm
Accessed on January 16, 2011

7. http://www.eng.umd.edu/~nsw/ench485/lab9d.htm
Accessed on January 16, 2011
-10
0
10
20
0 20 40 60 80 100
C
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
m
L
)
Temerature (C)
Hydrolyzed Sucrose Concentration-
Temperature