DETERMINATION OF ENZYME ACTIVITY THROUGH GLUCOSE USING

DINITROSALICYCLIC COLORIMETRIC METHOD WITH THE EFFECT OF

TEMPERATURE
Krizzia Perez
Krystal Reformado
Angel Reyes
Angela Roque
Joshua Santos

ABSTRACT
Enzyme activity varies due to different physical and chemical factors. In the experiment, saturated
picric acid colorimetric method was used to determine the maximum capacity of invertase activity in
increasing concentration of the standard and through a graphical representation, the “best fit” straight
line was determined. The invertase was subjected to different temperatures as to establish the effects
on its activity through a graph.

against either pH or temperature, the

INTRODUCTION curve usually has a peak or the optimum.
Living cells is the site of tremendous
biological activity called metabolism. This The region of optimum temperature for
is the process of chemical and physical activity is not necessarily at or near the
change which goes on continually in the temperature values normal for the living
living organism. Tissue repair, conversion cell from which the enzyme was taken.
of food to energy, excretion of waste Not even the region of optimum pH for
material and reproduction are the activity necessarily at or near the region
activities that are associated with life and of the optimum pH of for stability, which is
majority of these biological activity are not also the same as to temperature.
spontaneous.
Catalysis makes these possible which is
necessary in all life form. 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.
Enzymes are all proteins, simple or
conjugated. Most of the globular proteins
are involved in metabolic functions. They
are high molecular weight compounds made
up principally of chains of amino acids linked
together by peptide bonds. They all share
the same property of protein. They are
antigenic. They are denatured by such
Figure 1 Enzyme Activity - Graphical
agents as elevated temperature and Presentation of Catalyzed and Uncatalyzed
extreme pH values. Their physical state Reaction
and catalytic function depend distinctly
upon a number of physical factors such as
pH, temperature, and ionic strength. All Activation energy and its relationship to
enzymes are functionally specific to the free energy charge of a reaction can
varying degrees. be best shown in a graphically.
Enzyme activity is the rate of the In the experiment, Invertase was the used
catalyzed reaction. When it is plotted enzyme. Invertase is an enzyme that
catalyses the hydrolysis of sucrose in
which the bonds of the sugar splits into
two, glucose and fructose. It belongs to a
class of enzymes known as glycosidases.
Some of these enzymes split the bonds
while others twist the bonds at the same
time. The term “invertase” refers to which
enzyme was from, either fungal, bacterial
or plant.
Glucose was used as the substrate of this
experiment in which the enzyme acts to.
Glucose 𝐶6𝐻12𝑂6 is a mosaccacharide sugar
which the product of photosynthesis in
plants. It is the source of energy in the
cell function from the circulating free
sugar in blood and a major participant in
metabolism. Glucose and fructose make
up sucrose.
Figure 2 Chemical structure of glucose
Glucose units in long chains make up
polysaccharides. Glucose is commonly
used in foods, medicines, brewing, and
wine making as the source of various
other organic chemicals. It is also known
as D-glucose, D-glucopyranose, grape
sugar, corn sugar, dextrose and cerelose.
Figure 3 Chemical structure of Dinitrosalicyclic
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 to
form 3-amino-5-nitrosalicylic acid, which
absorbs light strongly at 540 nm. It was
first introduced as a method to detect
reducing substances in urine and has
since been widely used, for example, for
quantificating 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. In determining the enzyme
activity of the invertase through glucose,
this experiment aims to extract invertase
from the Baker’s yeast and to determine
the effects of changes in pH and
temperature on the reaction rates of an
enzyme-catalyzed reaction.
EXPERIMENTAL
1. Surcose Assay Using
Saturated Picric Acid Colorimetric
Method
A set of test tubes were prepared by
following the table below. (See Table 1)
All test tubes were incubated in 90°C
water bath for five (5) minutes. A 3 drops
of Concentrated Hydrochloric acid then a .
5mL of Potassium Hydroxide was added to
the solution to neutralize the acidity
resulted when hydrochloric acid was
added then a 2.80 mL of 0.1 M buffer
solution, pH 5 was added and finally a 3
mL of Dinitrosalicylic was added; the test
tubes were incubated for 10 minutes to
develop the characteristic red brown color
at 95 degrees Celsius. The test tubes were
subjected to spectrophotometer at 540nm
molar absorbance. The absorbance of test
tubes #’s 1 to 6 was presented in a
graphical presentation for analysis.
Table 1 Test tube preparation for Glucose Assay using Saturated Picirc Acid Colorimetric Method.
Test Tube No. Blank 1 2 3 4 5 6

Sucrose Standard Solution 0.0 0.2 .50 .75 1.0 1.2 1.50
𝟏𝒎𝒈𝟏𝒎𝒍 5 5

Distilled Water 1.5 1.2 1.0 .75 .50 0.2 0.0

5 5

curve constructed in the DNS colometric

Effect of Temperature on Invertase method.
Activity
Different temperature of water bath set- RESULTS AND DISCUSSION
ups was prepared. (See Table 3) A set of A. Glucose Assay Using
six (6) test tubes containing 1.50 ml Saturated Picric Acid Colorimetric
sucrose solution was prepared. Method

Table 3 Test tube preparation for Effect of Table 4 Data of Glucose Assay using Saturated
Temperature Invertase Activity Picric Acid Colorimetric Method
Test Tube 1 2 3 4 Test Amount of Acid- A540
No. tube hydrolyized Surose
Temperatur 20° 30° 50° 60°C no. (mg/ml)
e C C C

1 0.25 0.078
Test Tube 5 6
2 0.50 0.084
No.
3 0.75 0.096
Temperature 70°C 90°C
4 1.00 0.097
5 1.25 0.102
6 1.50 1.266
Each test tube was incubated separately
in its respective water bath set-up for five
Glucose, and other reducing sugars,
(5) minutes. A blank test tube for each of
reacts with DNS by oxidation reaction
the six (6) test tubes was prepared. The
producing a colored compound that shows
blank test tubes containing 0.8 ml
a maximal molar extinction at 530nm.
invertase and a 19.2 ml 0.1Mbuffer
Glucose is reduced into gluconic acid,
solution, pH5 was prepared. A 3.0 ml
same as to dinitrosalicylic acid to 3-amino,
dilute enzyme solution was added to the
5-nitro saliccycli acid,giving the
six (6) test tubes then incubated for
characterisctic red-brown color. Oxidation
another five (5) minutes. A 3.0 ml DNS
is a reversible chemical reaction in which
reagent and these were subjected to
one of the reactions is oxidation and the
spectrophotometer at 540nm molar
reverse reaction is reduction. Since the
absorbance and a blank test tube was
absorbance at 540 nm is linearly
prepared same as the preparation for the
dependent on the concentration or mass
6 test tubes except that denatured
of glucose the reaction can be used for
enzyme was used instead of enzyme stock
quantification of reducing sugars.
solution. A graphical representation was
In an enzyme assay, in order to be valid,
prepared for analyzation. And then
it must satisfy at least three conditions:
determine the amount of sucrose
(1) Activity must be proportional to the
hydrolyzed using the sucrose standard
amount of enzyme source added; (2)
activity must be constant during the time
period of the enzyme; and (3) assay must
be carried out at saturating substance
concentration.
During an enzyme-catalyzed reaction, in
the experiment is invertase was used, the
enzyme binds to a substrate to form a
complex. Formation of complex leads to
formation of transition-states species,
which then forms the product. All the
invertase, glucose and reagents were
allowed to react in the process to be
subjected in reading its absorbance or
molar extinction.
Figure 4 Simplest Kinetic Equation - Michaelis-
Menten Equation
Absorbance was measured through the
use of spectrophotometer which measures
the amount of light transmitted through a
sample at a given wavelength. The read
absorbance of standard test tube was
subtracted to blank test tube. This will
give only the absorbance of the invertase,
without the additional reagents.
To graph, the x-axis is the concentration
of acid-hydrolyzed sucrose in milligram
over milliliter (mg/ml) and the y-axis is
the absorbance. The graph (Fig. 5) shows
a straight line, the standard’s best fit line
and the plotted absorbance of each test
tube. Though the accumulated data, the
plotted absorbance are near each other
and there is a linear trend in the original
process in which is in test tube number 5,
there is always be one in the finite block
of data taken that will represent it. The
possible faults in sucrose assay were (1)
inappropriate measurement of reagents
and wrong procedure; (2) DNS was not
reactive; and (3) technical deficiencies of
the used spectrophotometer.
The “best fit” line of the standard shows
the increasing enzyme activity in
increasing concentration of the standard.
This follows the principles of the Law of
Mass Action. The binding of the substrate
to the enzyme occurs because of the high
specific interaction. In some cases, when
the enzymes are occupied already by the
substrate, the graph will orient a
downward slope indicating that the
enzymes cannot bind the excess
substrate. The calculated slope-intercept
form using Microsoft Excel application is y
= 5.445x R² = -87.8.
The graphical presentation for the glucose
assay using saturated picric acid method
is found in the next page.
B. Effect of pH and
Temperature on Invertase Activity
The optimum temperature of the invertase
should be at 60°C (140°F). For the
invertase to be exposed in elevated
temperature and extreme pH after
reaching its optimum, the enzyme will
slope down indicating denaturation of
enzyme. It indicates that an enzyme’s
reactivity is controlled and occurs to a
limited extent in the biological reactions.
In the experiment, the graphs did not
meet the expected orientation which is
bell shaped graph. It infers that the
activation energy is spontaneous that the
transition state was inferred to the highest
value of absorbance in the graph. The
experiment had limitations of
experimental technique. The effectivity of
the invertase was uncertain. The assumed
optimum temperature has the highest
value of absorbance which is 60°C,
respectively. The test has shown that the
optimum temperature was 60 degrees
Celsius for enzymatic activity. The highest
peak of the curve represents the highest
concentration which was determined by
the Absorbance(Fig 6).
Table 5 Data of Effect of Temperature on 20 .059 0.140
Invertase Activity
Temperature Amount of A540 30 .062 0.
(°C) Acid- 079
hydrolyized 50 .115 0.107
Surose 60 .280 0.174
(mg/ml) 70 .150 0.140
90 .065 0.097

Sucrose Standard Curve

0.8
f(x) = 5.45 x
0.7 R² = 0.69
0.6
0.5
0.4
Absorbance, 540 nm Linear ()
0.3
0.2
0.1
0
0 0.02 0.040.060.08 0.1 0.120.14 0.160.18
Concentration, mg/mL

Figure 5 Standard "Best Fit" Straight Line of Sucrose Assay using DNS reagent.
 Effect of Temperature on Enzyme Activity
0.3

0.25

0.2

0.15
Concentration, mg/mL
0.1

0.05

0
10 20 30 40 50 60 70 80 90 100
Temperature, Celsius

Figure 6 Graph of Effect of Temperature on Enzyme Activities

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