ENZYMES: EFFECT OF CHANGES IN pH WITH INVERTASE ACTIVITY

Maria Andrea Dagala, Elisha de Guzman, Katrina de Lara,

Jesmae Deiparine, Jorina Diaz, Doxa Espina
Group 3 2A Medical Technology Biochemistry Laboratory

ABSTRACT
INTRODUCTION
Enzymes are catalytic proteins found in a living
organism that hasten reactions by lowering the
activation energy of the reaction, as seen in the
figure below.

as it splits the bond between glucose and

fructose as it adds hydrogen and hydroxide,
separating the two types of sugar.[5] Sucrose or
table sugar (C12H22O11) is a white crystalline
solid, soluble in water, sweet in taste and is
dextro-rotatory. As said above, Sucrose is
hydrolyzed in equal amounts of glucose and
fructose by treatment of dilute acid or invertase
that will change dextrorotatory to levorotatory.
This conversion of optical rotation is called
inversion. And the products formed are called
invert sugars. [10]

Figure 1: Comparison of an uncatalyzed and

catalyzed reaction.[9]
These enzymes would not be consumed, they
would be then ready again for the next reaction.
Without these, biologic functions would take far
long, enzymes hastens the rate of reaction by
10^6- 10^12 times to make life possible .[6][7][3][4]
Enzymes are globular protein, they are
susceptible to changes in pH and temperature
that can cause an alteration to the secondary and
tertiary structure of the enzyme. Most enzymes
are in need of cofactors or coenzymes, that can
take form as vitamins (organic molecules such as
Vitamin C, Vitamin K) or minerals (mostly
positive-charged metals) that helps in the
enzyme-substrate binding.
A
complete
active
enzyme
with
its
coenzyme/cofactor is called a holoenzyme. If
there is no coenzyme/cofactor present, it is called
as an apoenzyme. As said above, enzymes are
affected by changes in pH and temperature due
to their globular nature. Increase in temperature
and pH goes in the same way, as either of them
increases, this would heighten enzymatic activity
upto to maximal efficacy when it reaches the
point of optimal pH or temperature, going beyond
that would cause denaturation or unfolding of the
enzyme. [7][12]
In this experiment, the enzyme used was an
invertase extracted from yeast. Invertase, or
beta-fructofuranosidase has the ability to
break sucrose, a disaccharide, down into the
simple sugars glucose and fructose by Hydrolysis

Figure 2: Hydrolysis of Sucrose by the enzyme

invertase.[8]
This
experiment
entitled
the
use
3,5
Dinitrosalicylic acid (C7H4N2O7) also known as
DNS or DNSA, IUPAC name 2-hydroxy-3,5dinitrobenzoic acid.
DNSA is used in colorimetric determination of
reducing sugars and to analyze glycosidase
activity by quantitation of enzymatically released
reducing
sugar.
Dinitrosalicylic
colorimetric
method is used to test the presence of free
carbonyl functional group (C=O), termed as the
reducing sugar, where the oxation of the
aldehyde functional group is also involved. [11]
The objectives of the experiment are to monitor
the
enzymatic activity
of glucose using
dinitrosalicylic colorimetric method, to extract
invertase from Bakers yeast and to determine
the effects of changes in pH and to know what
would be the optimum pH for maximal invertase
enzyme activity.
EXPERIMENTAL
A. Samples Used
Standard used:
Sucrose standard solution, 100 mg/L
Reagent use:
Dinitrosalicylic acid (DNS) reagent
Solution used:
Sucrose solution, 10 g/L

B. Procedure
1. Preparation of Denatured Invertase
Stock Solution
a. In boiling hot water, 100 mL of enzyme stock
solution was incubated for 10 minutes and the
solution was allowed to cool.
b. If frothing occurred, only the supernatant was
collected and served as the denatured enzyme
stock solution.
2. Sucrose Assay Using Dinitrosalicylic
Colorimetric Method
a. A series of test tubes were prepared as:
Table 1. Sucrose Assay Test Tubes
Tube No.
blank (1)
2
3
4
5
6
7

mL sucrose std
solution
0
0.25
0.50
0.75
1.00
1.25
1.50

mL distilled
water
4.50
4.25
4.00
3.75
3.50
3.25
3.00

b. Each test tubes were added with 3 drops of

concentrated HCl tube and mixed well. It was
incubated at 90C water bath for 5 minutes.
c. To neutralize the solution, 0.15 mL of 0.5 M
KOH was added.
d. 2.80 mL 0.1 M buffer solution at pH 5 was
added and mixed well.
e. 3 mL of DNS reagent was added. The test
tubes were then immersed in 95C water bath for
10 minutes until a formation of red-brown
coloration.
f. The absorbance at 540 nm was measured and
a standard curve was constructed.
3. Effect of pH on Invertase Activity
a. Three test tubes were prepared and added
with 2.9 mL of the prepared buffer solutions.

e. Blank solutions were prepared using

procedures a to d, without the sucrose solution.
f. The absorbance at 540 nm was measured and
the
amount
of
sucrose
hydrolyzed
was
determined
using
the
hydrolyzed-sucrose
standard curve in the dinitrosalicylic colorimetric
method.
4. Effect of Temperature on Invertase
Activity
a. Water baths were prepared in the following
temperatures: 30 (room temperature), 60 and
100 C.
b. 3 test tubes were prepared, each containing
1.5 mL sucrose solution and heated for 5 minutes
in each water bath.
c. In another test tube, 0.80 mL enzyme stock
solution was diluted with 19.20 mL 0.1 M buffer
solution, pH 5.
d. Each test tube was added with 3 mL dilute
enzyme solution. It was then heated for another
5 minutes.
e. 3 mL of DNS reagent was then added and
immersed in 95C water for 10 minutes until a
red-brown color was developed.
f. Blanks were prepared by following procedures
a-e, without the sucrose.
g. The absorbance at 540 nm was measured and
the
amount
of
sucrose
hydrolyzed
was
determined using the sucrose standard curve in
the dinitrosalicylic colorimetric method.

RESULTS AND DISCUSSION

The absorbances obtained using buffers with pH
of 2.2, 4.6 and 7.4 were 0.028nm, -0.003nm and
-0.001nm respectively. The concentrations of the
invert sugar were computed using the equation
derived from the standard curve.

Table 2. Test Tubes for Effect of pH on Invertase

Activity
Tube No.

pH of Buffer solution

2.2

4.6

7.4

b. Each test tube was then added with 0.10 mL

enzyme stock solution. It was heated in 60C
water bath for 5 minutes.
c. The mixture was added 1.50 mL of sucrose
solution and heated in 60C water bath for 5
minutes.
d. 3 mL of DNS was then added and heated in
95C water bath for 10 minutes.

Figure 3. Acid hydrolyzed sucrose standard

curve
y = 6.6939x
0.028nm = 6.6939 x

x = 0.028 / 6.6939
x = 0.00418
-0.003nm = 6.6939 x
x = -0.003 / 6.6939
x = 0.000448
-0.001nm = 6.6939 x
x = -0.001 / 6.6939
x = 0.000149

function. When the pH deviates from the ideal

conditions, enzyme activity will slow down and
eventually stop, depending on how much the
conditions are altered in the enzyme. Depending
on the enzyme and how extreme the pH change
gets, these changes may permanently "break"
the enzyme or the enzyme may return to normal
once conditions get back to the enzyme's ideal
range.

Changes in pH would cause change in shape of

an enzyme [2]. A change in the structure of the
enzyme affects the rate of the reaction. Not only
on enzymes, the pH levels may also affect the
shape and charge properties of the substrate.
shapes determine their function, so
pH
Amount
of
Acid- Absorbance Enzymes'
@
changing the shape around can impair the
Hydrolyzed
Sucrose 540nm
enzyme's function, preventing it from speeding
(mg/mL)
up chemical reactions
2.2
0.00418
0.028
4.6
0.000448
-0.003
Within a narrow pH range, changes in the
7.4
0.000149
-0.001
structural shapes of the enzymes and substrates
may be reversible. But for a significant change in
pH levels, the enzyme and the substrate may
undergo denaturation. In such cases, they cannot
The values of the pH and concentration of invert
identify each other. Consequently, there will be no
sugar were plotted. The graph obtained from the
reaction that will take place. This is the reason
experiment not the expected bell-shaped graph.
pH affect enzyme activity. As for invertase, a
great increase or decrease in the optimum pH of
the enzyme would result in loss of activity in the
enzyme, or denaturation.
Table 3.Table of the pH of buffer used, amount
of acid hydrolyzed sucrose in (mg/mL) and
absorbance at 540nm

Figure 4. Graph of the Effect of pH on Invertase

Activity
It was expected that the pH that would show the
greatest activity will be the standard with buffer
of pH 4.6. However, the activity of invertase with
pH 4.6 and 7.4 are significantly much less
compared to the activity it had at pH of 2.2. The
optimum pH now, according to the results of the
experiment, is 2.2. This does not agree with the
expected optimum pH of 4.5, since the optimum
pH of invertase is 3.5 5.5 [1] and pH 4.6 falls in
that range.
All enzymes have an ideal pH, a pH wherein the
enzyme will perform most efficiently (assuming
other conditions are also ideal). However, each
enzyme has an ideal pH suited for its specific

A flawed outcome, which resulted in an

ambiguous optimum pH could be obtained from
human error. The standard curve derived was not
accurate as the values fluctuate significantly and
are not of increasing value. Some factors that
contribute to inconclusive results are the
following: The first one would be the inaccurate
temperatures used in heating the standards.
Since the temperature also affects enzymatic
activity. Another factor is the inaccurate and
imprecise volumes delivered for making the
standards. And also a positive result of a redbrown colored solution was also not obtained
upon addition of DNS to the invertase solution in
the experimentation. The machine itself can also
be one of the reasons why the absorbances that
came out were erratic. The cuvettes, pipettes and
test tubes used can also be contaminated that
lead to erroneous results

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