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Determination on the Effect of Temperature on Invertase Activity Using

Spectrophotometry
By: Prince Dario De Guzman5, Margarita Jai B. Cobangco1 Ernesto Rafael C. Dayrit2, Denise Victoria DG. De
Guzman3, and Mellanie Claire C. De Guzman4

Group 3, 2F Medical Technology, Faculty of Pharmacy, University of Santo Tomas, España, 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 2002). Extreme pH values result in loss of


Living cells is the site of tremendous activity for most enzymes. Furthermore, there is
biological activity called metabolism. This is a most favourable pH for enzyme – the point
the process of chemical and physical change where enzyme is most active. This point is
which goes on continually in the living known as the optimal pH.
organism. Tissue repair, conversion of food to There are different classification for
energy, excretion of wastes material and enzymes: Oxidases or Dehydrogenases for
reproduction are the activities that are Oxidation-reduction reactions, Transferases
associated with life and majority of these for Transfer of functional groups, Hydrolases
activities are not spontaneous. for Hydrolysis reactions, Lyases for Addition to
Catalysis makes these possible which is double bonds or its reverse, Isomerases for
necessary in all life forms. It is the acceleration Isomerization reactions, and Ligases or
of a chemical reaction through the presence of Synthetases Formation of bonds with ATP
some substances that in which itself cleavage
undergoes no permanent chemical change. Invertase, or beta-fructofuranosidase, is
The catalysts of biological reactions are an enzyme derived from yeast. It catalyzes the
enzymes. hydrolysis (breakdown) of sucrose, which is a
An enzyme is a biomolecule, usually a table sugar, down into the simple sugars
protein, that acts as a biological catalyst to glucose and fructose. The resulting product,
speed the rate of a biochemical reaction also known as inverted sugar syrup. Invertases
(Boyer, 2006) which has the following cleave the O-C(fructose) bond. Though the
characteristics: First, the basic function of an enzyme is inclined to higher levels of activity at
enzyme is to increase the rate of a reaction. low temperatures, it is best when used at about
Second, most enzymes act specifically with 60oC, because sucrose will split faster at higher
only one reactant (called a substrate) to temperatures. [3]
produce products. Third, enzymes are Baker's yeast is the common name for
regulated from a state of low activity to high the strains of yeast commonly used as a
activity and vice versa. [2] Enzymes are leavening agent in baking bread and bakery
affected by changes in pH (Campbell & Reece, products, where it converts the fermentable
sugars present in the dough into carbon enzyme stock solution with 19.20 mL 0.1 M
dioxide and ethanol. Baker's yeast is almost buffer solution at pH 5. After dilution, 3 mL of
always of the species Saccharomyces the diluted enzyme was added to each test tube
cerevisiae, followed by incubation for 5 minutes. After
3,5-Dinitrosalicylic acid (DNS or DNSA, incubating, 3 mL of DNS 9 3,5-dinitrosalicylic
IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) reagent was added to the test tubes,
acid) is an aromatic compound that reacts with followed by immersion of test tubes in 95°C
reducing sugars and other reducing molecules. water bath for 10 minutes in order to develop
It was first introduced as a method to detect the red brown color characteristic.
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

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


other sugar. Sucrose is the only non-
reducing common disaccharide. Consequently, The same procedure was done in the
test for sugar detection using DNS (3,5- second set of test tubes; the only difference is
dinitrosalicylic acid) solution result in negative that denatured enzyme was added instead of
readings for sucrose. However, this method enzyme stock solution.
can still be applied if sucrose is first hydrolyzed
Table 1-1. Components of Standard Set of Test
in an acid solution to yield glucose and Tubes
fructose. Test Temperature Enzyme stock Denatured
This experiment seeks to evaluate and tube (°C) solution enzyme

validate the optimum temperature of an 1 20 + -


invertase activity and analyze the possible 2 30 + -
outcomes by controlling different variables with 3 50 + -
the use sensitive technique like 4 60 + -
spectrophotometry and linear regression 5 70 + -
analysis. 6 90 + -
Materials and Methods
Series of water baths were prepared Table 1-2. Components of Blank Set of Test Tubes
with varied temperatures; 20, 30, 50, 60, 70, Test Temperature Enzyme stock Denatured
tube (°C) solution enzyme
and 90°C. Then, two sets of six test tubes
were prepared. For the first set which served 1 20 - +
as standard, 1.5 mL of sucrose solution was 2 30 - +
added followed by separate incubation in each 3 50 - +
water bath for 5 minutes. While incubating, a 4 60 - +
diluted enzyme stock solution was prepared in 5 70 - +
a separate test tube by mixing 0.80 mL 6 90 - +
rather large in order produce sufficient reducing
After the cooling process, all solutions sugar for color development that would still be
were transferred to separate cuvettes. The detectable. This method tests for the presence
absorbance was measured by using a of free carbonyl group (C=O), the so-called
spectrophotometer with 540 nm wavelength. reducing sugars. This involves the oxidation of
The absorbance of the blank solution was put the aldehyde functional group present in, for
to zero. The resulting absorbances were example, glucose and the ketone functional
tabulated and the concentration of each group in fructose. Simultaneously, 3,5-
hydrolyzed sucrose was determined using the dinitrosalicylic acid (DNS) is reduced to 3-
standard curve constructed in the amino,5-nitrosalicylic acid under alkaline
dinitrosalicylic colometric method. All the data conditions.
collected were tabulated and interpreted using One of the factors in the stability of
figures and tables. enzymes is temperature. The optimum
temperature of the invertase activity should be
Results and Discussion at exactly 60°C (140°F). If the invertase was
A stock solution is a concentrated exposed in elevated temperature and extreme
solution to be diluted to some lower pH after reaching its optimum, the enzyme will
concentration, whereas, denatured enzyme slope down indicating denaturation of enzyme.
have distorted active sites therefore disabling This phenomenon indicates that enzymes
the enzyme to lock up with a substrate. reactivity is controlled and occurs to a limited
Denaturing an enzyme was done by subjecting extent in the biological reactions.
it to high temperature where they vibrate
vigorously that the bonds holding the protein Table 2-1. Absorbance obtained
structure in its specific shape becomes broken. Temperature Absorbance540
(°C)
20 0.33
30 0.216
50 -.026
60 0.678
70 0.410
90 -.124

Figure 3-1. Graph of Absorbance vs. Temperature


Figure 2. Hydrolysis of Sucrose into Glucose and
Fructose by invertase.
Based on the data presented, the group
Sucrose is made up of glucose and got a negative value of absorbance in test tube
fructose and is connected by glycosidic 3 and 6 with temperatures of 50 and 90°C.
linkage. Upon hydrolysis the glycosidic bond is However, a bell-shaped curve was still
broken and results in formation of glucose and obtained. Theoretically speaking, the linear
fructose. The hydrolysis reaction takes place sequence must continue on raising until it
at slower rate, but upon addition of enzyme reaches the peak or the optimum temperature.
invertase the reaction takes place at rapid rate. As the temperature continue to increase
Dinitrosalicylic acid reacts with reducing surpassing the optimum value which is 60°C,
sugars and other reducing moleculesto form 3- the linear sequence will start to drop indicating
amino-5-nitrosalicylic acid, which absorbs light the start of denaturation of the enzyme.
strongly at 540 nm thus the amount of enzyme There are many possibilities of having a
in the reaction mixture therefore had to be negative absorbance; one is that the samples
might have been contaminated due to
improper cleaning of laboratory apparatus Table 2-3. Derived Concentration from
such as test tubes, cuvettes, and pipettes. The Test Temperature HSC Absorbance 540 nm
Tube (°C) (mg/mL)
Dilution process might not have been
1 20 7.67 0.33
successfully done because of human error in
measuring the specific volume. Also, the group 2 30 5.02 0.216
had a limited time in performing the 3 50 -0.604 -.026
experiment thus the needed temperature for
4 60 15.8 0.678
each water bath has not been successfully
provided. 5 70 9.53 0.410
The concentration of the hydrolyzed 6 90 -2.88 -.124
sucrose under different temperatures has been
determined by the analysis in the result of the calibration curve
Sucrose assay using dinitrosalicylic colometric
method from the other group. By substituting the specific absorbance
to the y-value of the calibration curve divided by
Table 2-2. Conc. And Absorbance of HSC from DNS the value of 0.043, the group has derived the
Colorimetric Method specific concentration which is x.
Test Amount of Hydrolyzed Absorbance540
Tub Sucrose (mg/mL)
e
Blan 0 0
k

x=y
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
"which is
the
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. (C2) In the
table is derived from the dilution formula
wherein C1=10mg/mL, V1= (specific volume of
sucrose standard solution), and V2= total
specific
absorbanc
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.

e"0.043
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.
2. Boyer, Rodney. (2006). Concepts in Biochemistry (3rd ed.)
John Wiley and Sons Asia Pte Ltd. 131- 155

Figure 3-3. Graph of Derived Conc. vs. Temperature 3. Campbell, M.K. & Farell, S.O. (2009). Biochemistry (6th ed.)
Philippines: Cengage Learning Asia Pte Ltd. 143- 150

This figure above proved that the 4. Mckee, J.R. & Mckee, T. (2009). Biochemistry: The
derived concentration from the calibration Molecular Basis of Life (4th ed.). New York: Oxford University
Press. 184- 195, 202- 219
curve is correct due to the bell-like orientation
5. Reiner, J.M. (1969). Behavior of enzyme Systems. New
formed by the linear sequence of the graph. York: Van Nostrand Reinhold. 45- 54, 113- 116, 261- 284.

From online websites and researches:


References
From books: 6. http://www.eng.umd.edu/~nsw/ench485/lab14.htm
Accessed on January 16, 2011
1. Atkins, Robert C. & Francis A.Carey. (2002). Organic
Chemistry: A Brief Course (3rd ed.). Mcgraw-Hill 7. http://www.eng.umd.edu/~nsw/ench485/lab9d.htm
Companies,inc. USA,NY pp310,313 Accessed on January 16, 2011