Dinitrosalycylic Colorimetric Method for Sucrose Assay and the Effects of pH Change in

the Activity of Enzymes

Chua, K.M.D., Cocjin, C.M.PH.R, Dizon, J.A.H., Donato, L.P.G., Dumaplin, R.A.L, Francisco,
R.A.2B-PH, Group 3, Department of Pharmacy, Faculty of Pharmacy, University of Santo
Tomas, Espaa Boulevard, 1015 Manila, Philippines
ABSTRACT

Living organisms are composed of intricately coordinated systems of chemical reactions. Most of
these chemical reactions are catalyzed by enzymes that allow these reactions to proceed at a rate
sufficient to sustain life.[1] Enzymes are very efficient catalysts for biochemical reactions. They
speed up reactions by providing an alternative reaction pathway of lower activation energy.
Enzymes are very efficient catalysts for biochemical reactions. They speed up reactions by
providing an alternative reaction pathway of lower activation energy.[2] The performance of an
enzyme depends on various factors, such as temperature, pH, cofactors, activators and inhibitors.
The 3,5-dinitrosalicylic acid or 3,5-DNS is used in the DNS Colorimetric Method, which
involves the oxidation of the aldehyde functional group present in, for example, glucose and the
ketone functional group in fructose.[5] 3,5-DNS also has many uses in the medical field. 3,5-DNS
in fact was introduced first as a method in detecting reduced substances in urine and also used
for quantifying carbohydrate levels in blood. [6] As stated earlier, the performance of an enzyme
depends on various factors, such as temperature, pH, cofactors, activators and inhibitors.
Enzymes are proteins, meaning, enzymes are also very sensitive to pH changes. The reaction is
most active when its pH is at its peak or what we call optimum pH. Five test tubes were used
as vessels for the hydrolyzed sucrose. Apart from the five test tubes, six test tubes were also used
in testing the effects of pH on enzymatic activity. The UV-Vis Spectrophotometer was used to
measure the absorption of the samples of the test. To compute for the concentration, the
formula Invertase concentration = (ANET- y-intercept) divided by the value of the slope was used.
To graphically represent the relation of concentration and absorption, a Standard Calibration
Curve was devised. A bell-shaped curve was also observed between the relationship of the pH
and invertase concentration.
INTRODUCTION
Living organisms are composed of
intricately coordinated systems of chemical
reactions. Most of these chemical reactions
are catalyzed by enzymes that allow these
reactions to proceed at a rate sufficient to
sustain life.[1] Enzymes are very efficient
catalysts for biochemical reactions. They
speed up reactions by providing an
alternative reaction pathway of lower
activation energy. Enzymes are very

efficient catalysts for biochemical reactions.

They speed up reactions by providing an
alternative reaction pathway of lower
activation energy.[2] The performance of an
enzyme depends on various factors, such as
temperature, pH, cofactors, activators and
inhibitors.
Sucrose or table sugar is obtained from
sugar cane or sugar beets. Sucrose is made
from glucose and fructose units. Sucrose is
hydrolyzed when it is treated with sucrose or

invertase. It forms a 1:1 mixture of glucose

and fructose. Glucose and fructose are called
invert sugars because the angle of the
specific rotation (Dextro-rotation to levorotation) changes from a positive to a
negative value due to the presence of the
optical isomers of the mixture of glucose
and fructose sugars.[3]
Invertase,
also
named
as
betafructofranosidase or sucrose, hydrolyzes
sucrose giving off glucose and fructose
separately. Specifically, the change or
inversion is done by the hydrolysis of the

terminal non-reducing beta-fructofuranoside

residues in beta-fructofuranosides.[4]

Figure 1: Chemical structure of 3,5dinitrosalicylic acid.

The 3,5-dinitrosalicylic acid or 3,5-DNS is
used in the DNS Colorimetric Method,
which involves the oxidation of the aldehyde
functional group present in, for example,
glucose and the ketone functional group in
fructose.[5] 3,5-DNS also has many uses in
the medical field. 3,5-DNS in fact was
introduced first as a method in detecting
reduced substances in urine and also used

for quantifying carbohydrate levels in blood.

[6]

As stated earlier, the performance of an

enzyme depends on various factors, such as
temperature, pH, cofactors, activators and
inhibitors. Enzymes are proteins, meaning,
enzymes are also very sensitive to pH
changes. The reaction is most active when
its pH is at its peak or what we call optimum
pH. The result of the effect of pH varies on a
combination of factors like the binding of an
enzyme to a substrate, the catalytic activity
of the enzyme, the ionization of the
substrate, and the variation of protein
structure. Figure 2, which is shown below,
depicts a perfect bell-shaped curve where an
enzyme reaction to pH is seen.[7]

Figure 2: pH and enzyme reaction

relationship
METHODOLOGY
In extracting invertase from yeast, we first
dissolved 0.25g bakers yeast in distilled
water to make a 250-mL solution. Then, we
allowed the solution to stand for 20 minutes
at room temperature. Lastly, the supernatant
will only be collected if sedimentation
occurs.
In preparing denatured invertase stock
solution, first, we incubated 100mL enzyme

stock solution in a boiling water bath for 10

minutes. Then, we allowed the solution to
cool. Lastly, the supernatant will only be
collected if frothing occurs.
For the Dinitrosalicylic Colorimetric method
for sucrose assay, a series of test tubes were
prepared.

Tube
No.

Blank

STD 1

STD 2

STD 3

STD 4

mL
sucrose
standard
solution
mL
distilled
water

0.10

0.50

1.00

1.5

1.50

1.40

1.00

0.50

Table 1. Measurements of Standards in the

Sucrose Assay
Then, 3 drops of concentrated HCl was
added to the test tube and was mixed well.
The test tube with the solution was
incubated in a 90oC water bath for 5
minutes. Then, 0.15 mL of 0.5 KOH was
added to neutralize the solution. Next, 2.80
mL of 0.1 M buffer solution, at pH 5 was
added and mixed well. 3mL of DNS reagent
was added. The test tubes were immersed in
a 95oC water bath for 10 minutes to develop
the red-brown solution. After cooling, the
absorbance was measured at 540 nm
wavelength. The hydrolyzed- sucrose
standard curve by plotting A540 against
concentration.
In measuring the effect of pH in enzyme
activity, we prepared first 6 test tubes and
were labeled with its respective pH values:
2, 3, 4, 5, 7, and 10. 0.10mL enzyme stock
solution was added to each test tube and was
thoroughly mixed. The test tubes were

incubated in a 60oC water bath for 5

minutes. Then, 1.50mL sucrose solution was
added to the test tubes and the mixture was
yet again incubated in a 60oC water bath for
5 minutes. 3mL of DNS reagent was added.
The test tubes were immersed in a 95oC
water bath for 10 minutes to develop the
red-brown solution. Then, the solutions were
cooled down. Next, blank solutions were
prepared based on the previous steps but this
time, denataured enzyme was added instead
of enzyme stock solution. Lastly, the
absorbance at 540nm was measured and
amount of sucrose hydrolyzed standard
curve
was
constructed
using
the
dinitrosalycylic colorimetric method.
RESULTS AND DISCUSSION
Enzymes are very efficient catalysts for
biochemical reactions. They speed up
reactions by providing an alternative
reaction pathway of lower activation energy.
Enzymes are very efficient catalysts for
biochemical reactions. They speed up
reactions by providing an alternative
reaction pathway of lower activation energy.
[2]
The performance of an enzyme depends
on various factors, such as temperature, pH,
cofactors, activators and inhibitors.
To get the concentrations of the standards,
the formula C1V1=C2V2 was used. The table
below will represent the values that was
computed or the concentration of the
sucrose.
Concentratio Absorbance
n (mg/mL)
STD 1
6.67
0.427
STD 2
33.33
0.516
STD 3
66.67
0.398
STD 4
100
0.4385
Table 2. Sucrose Assay Data

The data from table 2 will be used to make

the standard curve graph for hydrolyzedsucrose with concentration as the x-axis and
the absorbance will represent the y-axis.
0.6
f(x) = 0.01x
R = 0.66

0.4

Absorbance

0.2
0

50

100

150

Concentration (mg/mL)

Figure 3. Standard Curve Graph for

hydrolyzed sucrose
Figure 3 displays the relationship between
the concentration and absorbance of the
standards. There is also a huge gap between
each point. This means each point has
minimal relationship which may be caused
by: inactivity of sucrose, DNS might not be
active or there was some problem with the
measurements
given
by
the
spectrophotometer.
Enzymes are proteins, meaning, enzymes
are also very sensitive to pH changes. The
reaction is most active when its pH is at its
peak or what we call optimum pH. The
result of the effect of pH varies on a
combination of factors like the binding of an
enzyme to a substrate, the catalytic activity
of the enzyme, the ionization of the
substrate, and the variation of protein
structure.
pH

2
3
4

Invertase
Concentratio
n (mg/mL)
1421.732
1447.659
1473.585

ANET

0.022
0.014
0.006

5
1551.365
-0.018
7
1515.716
-0.007
10
1437.936
0.017
Table 3. Invertase Activity Data
Table 3 displays the data that was calculated
using the y-intercept, the slope, the
absorbance values of denatured enzymes
and enzyme stock solutions. ANET was
calculated with the formula ANET = ES-DE
and invertase concentration was calculated
with the formula Invertase concentration =
(ANET- y-intercept) divided by the value of
the slope.
1600
1550
1500
1450
Invertase Concentration (mg/mL)
1400
1350
10
0 20

pH

Figure 4. Effect of pH in Invertase Activity

Figure 4 shows that the bell-shaped curve
formed from the plots. The optimum pH is 5
indicating that the enzyme reaction will be
at its most active state. It states that the
enzyme only works at low pH levels, this is
because as the pH changes, there will be
denaturation which will affect the shape of
the invertase along with its effectiveness. It
is evident and also shown in the figure that
as the pH rises, the enzyme displays
denaturation.
REFERENCES
[1] Crisostomo, A., Daya, M., Farrow, F.,
Gabona, M., Liu, M., Pena, G. . . (2010).
Enzymes. Laboratory Manual in General
Biochemistry. (pp. 41-43). Quezon City. C &
E Publishing.

[2] Anonymous (n.d.). Enzymes. Retrieved

from
http://www.rsc.org/Education/Teachers/Reso
urces/cfb/enzymes.htm#6

[5] Wang, N. (n.d.). Glucose assay by

dinitrosalicylic
Colorimetric
method.
Retrieved
from
http://eng.umd.edu/~nsw/ench485/lab4a.htm

[3] Ophardt, C. (n.d.) Sucrose. Retrieved

from
http://chemwiki.ucdavis.edu/Core/Biological
_Chemistry/Carbohydrates/Disaccharides/Su
crose

[6] Anonymous. (2014). 3,5-dinitrosalicylic

acid.
Retrieved
from
http://www.ebi.ac.uk/chebi/searchId.do?
chebiId=CHEBI%3A53648

[4] Wang, N. (n.d.). Experiment no. 14

Enzyme Kinetics of Invertase
via Initial Rate Determination. Retrieved
from
http://www.eng.umd.edu/~nsw/ench485/lab1
4.htm.

[7] Anonymous. (n.d.) Enzyme Activity.

Retrieved
from
http://www.rpi.edu/dept/chem-eng/BiotechEnviron/IMMOB/enzymeac.htm