The Measurement of the Activity of Invertase in Sucrose Solution of pH 8 and

pH 12 using Dinitrosalicylic (DNS) Assay Colorimetric Method

Palomares, R., Pfleider, A., Polintan A., Reyes R., Rivera A., Santos, P.
2I-Pharmacy, Faculty of Pharmacy, University of Santos Tomas

ABSTRACT
All living organisms perform biochemical reactions inside their body. To help
accelerate and achieve necessary reaction rates, enzymes catalyse these reactions such
as digestion and metabolism. Enzyme activity is highly selective to specific substrates and
reactions thus, several factors can affect optimal enzyme activity. In this experiment, the
effect of pH on invertase activity was investigated. Samples with mixture of phosphate
buffer, pH 8.0 and 12.0, and invertase in two respective micro test tubes were added
with sucrose. The mixtures were then subjected to water bath prior to the addition of
DNS reagent which produced a red-brown color after a boiling water bath. A blank set-
up was prepared using PB buffer with pH 5.0 and without the addition of invertase.
Reading for absorbance of the samples were done by using a spectrophotometer at 540
nm. Results show that invertase hydrolysed sucrose into reducing sugars, glucose and
fructose, with an absorbance of 0.238, 0.279 and 0.335 in pH 8, 12 and blank samples
respectively. Using the DNS Colorimetric Assay standard curve, the concentration of
reducing sugars was 1.4302x10-3 mol/L for pH 8 and 3.8534x10-3 mol/L for pH 12,
interpreting to enzyme activity which are not optimal nor of standard concentrations in
respective pH levels.
INTRODUCTION Intermediate can also be a reactant
producing an enzyme (Figure 1.)
Enzymes are biological molecules
that affect the speed of different
chemical reactions in all biological
systems and forms of life. They are
essential for having a role in aiding in
digestion and metabolism in the body.
Also, enzymes aid in breaking large
molecules into smaller molecules so that Figure 1. Substrate and reaction
it can be absorbed by the body. Since specificity of Enzymes.
enzymes are highly selective due to the
shapes of the molecules, they catalyze There are several roles of
specific reactions only. Enzymes bind enzymes inside the body and each part
with the molecules called substrates. of the body has a specific enzyme in it.
Substrates bind to, active site, the region One of these is the Invertase, also called
on the enzyme molecule (Castro, 2014). beta-fructofuranosidase and is produced
The product formed with an enzyme is and purified from baker’s yeast. Acting as
called the reaction intermediate. an immune booster, anti-oxidant,
antiseptic are the primary roles of
invertase in humans. (Kulshresthaa et
al., 2013). This enzyme aid in the
hydrolysis from sucrose to fructose and
glucose. Sucrose is composed of an
alpha-D-glucose molecule and a beta-D-
fructose molecule which bonds to alpha-
1,4-glycosidic bond and with the
presence of the enzyme, invertase, in the
bond is cleaved due to hydrolysis
resulting to mixture of glucose and
fructose, (Figure 2) wherein the mixture
is called invert sugar (Wang, n.d.).
Figure 2. Conversion of Sucrose to
Glucose & Fructose using Invertase
Enzyme.
Enzymes activity depends on
different factors, one of which is the
change in pH. Each of the enzymes has
an optimum pH for it to be stable. For
invertase, the optimum pH is 4.5.
Changes in pH affects the shape of the
enzyme and the charge properties of the
substrate. These changes would not let
the substrate bind to the active site. In
the experiment, the activity of invertase
was observed in solutions with pH 8 and
pH 12.
To monitor the enzyme activity,
Dinitrosalicylic acid (DNS) Assay method
is used. This method is the measurement
of the products formed by reducing
sugars by hydrolysis (McDonald, 2012).
The principle of the DNS method is to
detect the presence of carbonyl group of
reducing sugars. The aldehyde functional
group in glucose and the ketone
functional group in fructose are involved
in oxidation which produce a reddish
brown colored complex, 3,5-
Dinitrosalicylic acid is reduced to 3-
amino-5-nitrosalicylic acid (Figure 3) with
an absorbance maximum of 540 nm. The
objectives of this experiment are: to
determine the effects of changes in pH
on the reaction rates of enzyme-
catalyzed reactions and to measure the
enzyme activity using the Dintrosalicylic
acid (DNS) Assay method.
Figure 3. DNS Acid Reaction with
Glucose producing ANS Acid.
METHODOLOGY
The extraction of invertase from
yeast and the preparation of the stock
solution were not done by group and was
already prepared by the instructors prior
to the experiment.
Reducing Sugars Assay Using the
Dinitrosalicyclic (DNS) Colorimetric
Method
Six micro test tubes were initially
prepared by serial dilution, the 1st test
tube contained a mixture of 125 µL of
inverted sugar standard solution and 875
µL of distilled water, an aliquot of 500 µL
was then transferred to the 2nd test tube
containing 500 µL of distilled water, the
serial dilution was repeated up to the 6 th
test tube and the extra 500 µL was
discarded. A blank solution was also done
by placing 500 µL of distilled water to
 another test tube. Table 1 shows
the concentration of the 7 overall test
tubes prepared for the DNS Assay of the
Standard Curve.
100 µL of an acetate buffer with a
pH of 4.7 and 500 µL of DNS reagent was
added to the 6 serially diluted test tubes
and was then placed in a boiling water
bath for 10 minutes.

Subsequently, 250 µL of 0.3M of

sucrose was then added to each of the
micro test tubes (Figure 5.) and was
Table 1. Volume of Invert Sugar Std. again heated in a water bath 60ºC for 5
Solution and Distilled Water added with minutes.
Final Concentration of Sample
The mixture was cooled down in
an ice bath. After cooling down the
solution, 1000 µL of water was added
and mixed. The finished sample was
placed in a microplate and the
absorbance was determined at 540 nm
using a spectrophotometer.
Figure 5. Addition of 0.3 M Sucrose
Effect of pH on Invertase Activity
Before the addition of DNS
The group pipetted 125 µL of 0.1M reagent, the micro test tubes were cooled
buffer solutions with a pH of 8 and 12 and down in an ice bath in order to halt the
were put into two respective micro test reaction. (Figure 6.)
tubes. Then, 25 µL of the enzyme
solution and 250 µL of distilled water
were added to each of the micro test
tubes and were mixed thoroughly. These
micro test tubes were placed in a water
bath with a temperature of 60ºC for 5
minutes. (Figure 4.)

Figure 6. Cooling with Ice Bath

Afterwards, 250 µL of the DNS
reagent was added to both of these micro
test tubes. These were then placed in a
boiling water bath for 10 minutes. (Figure
7.) 1000 µL of distilled water was added
Figure 4. Water bath of micro test tubes
to the micro test tubes after being placed
in the water bath and were mixed 0.2135, b equal to 16.9228 and r2 equal
together well. to 0.9176.

Standard Curve for Dinitrosalicylic

(DNS) Colorimetric Method
2.5 2.16

Absorbance
2 1.4623
1.5 1.1227
1 0.4387
0.1677
0.5 y = 16.92x + 0.2138
0.0953 R² = 0.9176
0
0 0.05 0.1 0.15
Figure 7. Boiling Water Bath for DNS
Concentration
Assay
200 µL of samples prepared were Figure 9. Standard Curve for DNS
then micropipetted into the wells of the Colorimetric Assay.
microplate. The absorbance was then
read at 540 nm using a Figure 10 shows the microplate of
spectrophotometer. (Figure 8.) the prepared invertase enzyme at pH 8
and 12, as the microplate was subjected
in the spectophotometer the following
absorbance readings were obtained
(Table 2.)

Figure 8. Absorbance reading of the

samples
RESULTS AND DISCUSSION
Figure 9 shows the standard curve Figure 10. Microplate with Highlighted
for Dinitrosalicylic (DNS) Colorimetric Portions for pH 8 and pH 12.
Assay using a linear graph wherein the
The DNS reagent acted as a
concentration is found on the x-axis and
chromogen, giving the red-brown color
the absorbance on the y-axis. Using
due to the presence of free carbonyl
linear regression, the slope of the
groups which is the reducing sugar. The
equation for the DNS Colorimetric
oxidation of the functional groups
Method was determined to be y=16.92x
aldehyde and ketone, which is found in
+ 0.2138, with values of a equal to
glucose and fructose respectively, caused pH Mean Concentration
the reduction of the DNS reagent. Absorbance (mol/L)
2 0.364 8.8770x10-3
Micro Absor Absor Absor Mean 3 0.346 7.8133x10-3
plate bance bance bance Absor 5 0.285 4.2080x10-3
Well 1 2 3 bance
Assig 7 0.297 4.9172x10-3
nment 8 0.238 1.4302x10-3
(E7-9) 0.217 0.252 0.246 0.238 12 0.279 3.8534x10-3
pH 8 Blank 0.335 6.7151x10-3
(F7-9) 0.254 0.281 0.301 0.279
pH 12
Table 3. Mean Absorbance and
(G7-9) 0.342 0.322 0.341 0.335 Concentration of different pH levels
Blank
Table 2. Absorbance Readings for pH 8, pH vs. Concentration of
pH 12 and Blank. Sucrose Using Invertase as
The mean absorbance for pH 8, Enzyme
pH 12, and blank is shown to be 0.238, 0.008877
0.01
0.279 and 0.335, respectively. Using the 0.0078133
0.008
data and equation given from the Concentration
0.006 0.0049172
Standard Curve for DNS Colorimetric 0.004208 0.0038534
Assay shown on Figure 1 above, the 0.004
0.0014302
concentration of the solution for pH 8 and 0.002

pH 12 can be computed by the 0

substitution of the mean absorbance in
replacement for the y value of the
equation. The computed concentration
for pH 8 was 1.4302x10-3 mol/L while the
computed concentration for pH 12 was
3.8534x10-3mol/L.
In comparison with other pH
conditions from the other groups’ results,
Table 3 shows the data of absorbance
and concentration of different pH
measurements ranging from pH 2, 3, 5,
7, 8, and 12. The pH of 8 does not show
optimum results with regards to the
computed concentration as it yielded a
much lower value compared to the other
conditions presented. The pH of 12
somehow shows inconsistency in the
trend shown at Figure 11.
0 5 10 15
pH
Figure 11. Bell Curve of pH vs.
Concentration of Sucrose Using
Invertase as Enzyme
Based on the DNS standard curve,
the absorbance obtained colorimetrically
by a spectrophotometer is directly
proportional to the DNS reagent reacting
to the concentration of reducing sugars
present in the sample. Hence, higher
absorbance is correlated with higher
concentration of glucose and fructose
which interprets to higher Invertase
activity. The theoretical optimal pH of
invertase is near pH 4.5 and high activity
between pH 3.5 to 5.5 forming a bell
curve. However, as shown in Figure 11,
there were inconsistencies showing an
optimal enzyme activity around pH 2.0,
inconsistent with the expected results. A
bell curve was also not formed to show
the expected effect of different pH levels.
Theoretically, alkaline solutions, with pH
8.0 and 12.0, causes partial destruction
of cysteine residues which greatly
decreases enzyme activity. However,
invertase activity in pH 12.0 was greatly
inconsistent with the expected result as it
shows higher concentration of reducing
sugars in comparison to pH 8.0
Moreover, obtained concentration values
were not consistent with the expected
range concentration values in the
standard curve, which translates to less
optimal activity of the enzyme invertase
to sucrose in the experiment conducted.
Inconsistencies in the data obtained may
be due to human errors in the procedure
such as inaccuracy in measurements.
CONCLUSION
A pH affects the activity of an
enzyme in such that enzymes have a pH
range in which they perform optimally.
Having a highly acidic or alkaline
environment may render the enzyme
ineffective, hence buffers assist in
maintaining the optimum pH range for
the enzyme's activity.
Absorbance level is directly
proportional to the concentration of
reducing sugars reacting to the DNS
reagent. Therefore, higher absorbance is
correlated with higher invertase activity.
Invertase hydrolyzes sucrose into
reducing sugars, glucose and fructose,
which produces the yellow to orange or
red-brown color reaction with the DNS
reagent and measured in terms of
absorbance levels using a
spectrophotometer.
According to Corrales, K. et.
al.(n.d.), the optimum pH range for
invertase is 4.5. To correlate with our
experiment, pH 8 and 12 yielded a
negative result as the optimum pH
environment for invertase as shown in
Table 2 where the concentration-
absorbance of pH 8 is deemed as the
lowest among all pH values tested
against the DNS reagent together with
the invertase sample. As for pH 12, the
standard bell shape curve and expected
results was not achieved due to
inconsistencies in the absorbance and
concentration values.
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