DETERMINATION OF ENZYMATIC ACTIVITY BY

DINITROSALICYLIC COLORIMETRIC METHOD AND pH

Esclanda, Verna Mae F., Flores, Mary Camelle C., Galvez, Czarina M.,
Gamboa, Maureen Allysandra J., Go Imon, Karl Louis L., Gudio, Klaudine Renee T.

ABSTRACT
Enzyme activity is affected by several factors. When the enzyme changes in shape and structure, its rate of reaction is
varied. Examples of the things that alters the rate of reaction of the enzymatic activity are pH and addition of
chemicals or reagents. In this experiment, dinitrosalicylic acid colorimetric method and changing in pH were done to
determine the effects of physical and chemical factors to the invertase activity of sucrose. Negative absorbance were
observed after they were tested in the UV-Vis Spectrophotometer.

INTRODUCTION enzyme that it reaches the maximum reaction

Enzymes are proteinaceous catalysts, which
speed up the rate of a biochemical reaction. They
reduce the activation energy that is essential for
starting any type of chemical reaction. With a low
energy requirement for activation, the reaction
takes place faster. The overall performance of an
enzyme depends on various factors, such as
temperature, pH, cofactors, activators and
inhibitors.1
Since enzymes are proteins, they are very
sensitive to changes in pH. Each enzyme has its
own optimum range for pH where it will be most
active. This is the result of the effect of pH on a
combination of factors: (1) the binding of the
enzyme to substrate, (2) the catalytic activity of
the enzyme, (3) the ionization of the substrate,
and (4) the variation of protein structure.2 The
initial rates for many enzymatic reactions exhibit
bell-shaped curves as a function of pH as shown
in the example below.
rate of the enzymatic activity.
Dinitrosalicylic acid (D.N.S.A. or 3:5-
dinitrosalicylic acid) is a (yellow) reagent used to
determine sugar content especially glucose. The
DNS technique is employed in order to estimate
sugar present in the blood, in the cerebrospinal
fluid and in other human bodily fluids. This is also
effectively used in the handling of requirements
for diabetic clinics in hospital laboratories,
considering that only 10 minutes is enough for
the process to take place and the reagents are
stable, cheap and easily prepared. The amount of
blood sugar in the blood has metabolic
implications and is used to determine the
presence of blood sugar-related disorders such as
hyperglycemia. One good way to assess blood
sugar level is through the use of dinitrosalicylic
acid.3

Fig.2 Chemical structure of 3,5-dinitrosalicylic acid

Fig.1 Effect of pH on enzymatic activity

The best explanation for this bell-shaped
curve is the stability of enzymes during the
alteration of pH. Meaning, low pH results to slow
reaction rate but too high pH shows the same
result because of enzymatic loss. The peak of the
“bell” demonstrates the “best” pH suitable for the
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 the calibration curve by enhancing the intensity
conditions: of the developed color.4

METHODOLOGY
A. Sucrose Assay By Dinitrosalicylic
Colorimetric Method
A series of test tubes were prepared as
Because dissolved oxygen can interfere with seen in the table below (Table 1). Three
glucose oxidation, sulfite, which itself is not drops of concentrated HCl was added to
necessary for the color reaction, is added in the each tube. The tube was mixed then
reagent to absorb the dissolved oxygen. incubated in a 90oC water bath for 5
minutes. Then, 0.15 ml 0.5 M KOH was
The above reaction scheme shows that one added to neutralize the solution. A 0.1 M
mole of sugar will react with one mole of 3,5- buffer solution about 2.8 ml was then
dinitrosalicylic acid. However, it is suspected that added and mixed. Three ml of DNS
there are many side reactions, and the actual reagent was put in the mixture. The test
reaction stoichiometry is more complicated than tubes were afterward immersed in 95oC
that previously described. The type of side water bath for 10 minutes to develop the
reaction depends on the exact nature of the characteristic red-brown color. The test
reducing sugars. Different reducing sugars tubes were then cooled and their
generally yield different color intensities; thus, it absorbance were measured at 540 nm.
is necessary to calibrate for each sugar. In
addition to the oxidation of the carbonyl groups
in the sugar, other side reactions such as the
decomposition of sugar also competes for the
availability of 3,5-dinitrosalicylic acid. As a
consequence, carboxymethyl cellulose can affect

Table 1. Test Tube Preparation For Sucrose Assay Using Dinitrosalicylic Colorimetric Method

Test Tube No./ Blank 1 2 3 4 5 6

Volume
Sucrose Std. 0 ml 0.25 ml 0.50 ml 0.75 ml 1.00 ml 1.25 ml 1.50 ml
Solution
(1mg/ml)
Distilled Water 1.50 ml 1.25 ml 1.00 ml 0.75 ml 0.50 ml 0.25 ml 0 ml

B. Effect of pH on Invertase Activity

Six numbered test tubes were prepared. for another 5 minutes. Three ml of DNS
2.9 ml 0.1 M buffer solution with different reagent was then added. The test tubes
pH was added in each test tube: were immersed in 95oC water bath to
develop the characteristic red-brown
Table2. Test Tube Preparation For Effect of pH on color. The test tubes were cooled after.
Invertase Activity
Blank solutions were prepared by
Test
following the first steps but instead of
Tube 1 2 3 4 5
No. enzyme stock solution, denatured enzyme
pH 2 3 7 7.5 11 solution was added. The absorbance was
measured at 540 nm.
Enzyme stock solution with amount of
0.1 ml was added to each test tube. After
the test tubes were mixed, they were
incubated in 60oC water bath for 5
minutes. One and a half ml of sucrose
solution was added and the test tubes
were again incubated in 60oC water bath
RESULTS AND DISCUSSION

A. Sucrose Assay Using Dinitrosalicylic

Colorimetric Method
In the experiment, as the dinitrosalicylic
acid, a yellow dye, was incorporated in the
Table3. x and y Values for Amount of Acid-Hydrolyzed
test tubes with the presence of heat, the
Sucrose and Absorbance at 540nm
mixture slowly turned red-brown. This is Amount of Absorbance
because the conversion of the 3,5- Acid-Hydrolyzed at 540nm (y)
dinitrosalicylic acid to 3-amino-5-nitrosalicylic Sucrose (x)
acid, which contributed to the red-brown color 0 0
of the mixture. The DNS also reacted to 0.017 0.004
glucose, a product from invertase activity of 0.033 -0.001
sucrose, and converted to gluconic acid. The 0.050 -0.003
absorbance of each hydrolyzed sucrose on 0.067 -0.003
test tubes was identified using the UV-vis 0.250 0.048
spectrophotometer. 0.100 0.001

Fig.3 Breakdown of Sucrose to Glucose and Fructose

Through Invertase

To compute for the amount of acid-

hydrolyzed sucrose, the equation
was used. Using the formula, the following
were obtained:

Fig.4 Absorbance vs. Amount of Acid-Hydrolyzed Sucrose

The line drawn on the graph represented the

“best fit” line and was computed through the
linear regression function of a scientific
calculator. The slope-intercept form computed
was found to be y=0.215x-0.011.
In the graph shown for absorbance vs. amount
of acid hydrolyzed sucrose, a linear trend was not
identified. This is due to some possible causes.
First, inactivity of the sucrose if it was not freshly
prepared. Second, the dinitrosalicylic acid might
not be reactive. Or, the spectrophotometer was
defective or not sensitive enough and may
actually commit errors.

B. Effect of pH on Invertase Activity

y=absorbance
b=intercept
m=slope
 The intercept was found to be -0.011 while the
slope was 0.215. The following absorbance will
be used to compute for the concentration or
amount of acid-hydrolyzed sucrose:
Table4. Absorbance (at 540 nm) at certain pH
pH Absorbance at 540nm
2 0.036
3 -0.004
7 -0.013
7.5 -0.009
11 0.009
Invertase may be used over an extended pH
range with an optimum pH at 4.5. This enzyme is
fully active from pH 3.0 to 5.5. Use at pH values
over 6.0 is not recommended because it causes
denaturation of the enzyme.
The result of the experimental graph was very
far different from the ideal graph. The graph was
supposed to be bell shaped. But in this
experiment, the graph was U-shaped. This U-
shaped curve was due to the negative values of
the absorbance. This kind of error was committed
because of some possible factors like the pH of
the mixture was not accurate or the
spectrophotometer itself committed the errors.

REFERENCES
1
PH EFFECT ON ENZYMES. (n.d.).
Retrieved January 18, 2011, from
http://www.buzzle.com/articles/ph-effect-on-
enzymes.html
2
ENZYME ACTIVITY. (n.d.).
Retrieved January 18, 2011, from
http://www.rpi.edu/dept/chem-eng/Biotech-
Environ/IMMOB/enzymeac.htm
3
Tan, Sue Teresa. HOW TO USE DINIT-
ROSALICYLIC ACID
Retrieved January 18, 2011
http://www.ehow.com/how_5221277_use-
dinitrosalicylic-acid.html#ixzz1BIa2r4fx

4
Table5. x and y Values for Effect of pH on Invertase Activity Wang, N.S. GLUCOSE ASSAY
pH (x) Amount of Acid-Hydrolyzed BY DINITROSALICYLIC COLORIMETRIC METHOD
Sucrose (y) Retrieved January 18, 2011, from
2 0.218
http://www.eng.umd.edu/~nsw/ench485/lab4
3 0.033
7 -0.0093 a.htm
7.5 0.0093
11 0.09

Fig.5 Amount of Acid-Hydrolyzed Sucrose vs. pH