DETERMINATION OF ENZYME ACTIVITY THROUGH GLUCOSE USING PICRIC ACID

COLORIMETRIC METHOD, pH AND TEMPERATURE

Maria Feliza C. Abesamis, Marie Em Clarisse P. Acosta, Francheska M. Agustin,
Mary Christelle G. Aquitania, and Marulu Jane H. Bagsican
Group 1 2E Medical Technology Biochemistry Laboratory

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 pH and temperature as to establish the effects on its activity through a graph.

INTRODUCTION
Living cells is the site of tremendous biological
activity called metabolism. This is the process of
chemical and physical change which goes on
continually in the living organism. Tissue repair,
conversion of food to energy, excretion of waste
material and reproduction are the activities that
are associated with life and majority of these
biological activity are not spontaneous.
Catalysis makes these possible which is
necessary in all life form. It is the acceleration of
Figure 1 Enzyme Activity - Graphical Presentation of
a chemical reaction through the presence of
Catalyzed and Uncatalyzed Reaction
some substances that in which itself undergoes
no permanent chemical change. The catalysts of Activation energy and its relationship to the
biological reactions are enzymes. free energy charge of a reaction can be best
Enzymes are all proteins, simple or conjugated. shown in a graphically.
Most of the globular proteins are involved in In the experiment, Invertase was the used
metabolic functions. They are high molecular enzyme. Invertase is an enzyme that catalyses
weight compounds made up principally of chains of the hydrolysis of sucrose in which the bonds of
amino acids linked together by peptide bonds. They the sugar splits into two, glucose and fructose. It
all share the same property of protein. They are belongs to a class of enzymes known as
antigenic. They are denatured by such agents as glycosidases. Some of these enzymes split the
elevated temperature and extreme pH values. bonds while others twist the bonds at the same
Their physical state and catalytic function depend time. The term “invertase” refers to which
distinctly upon a number of physical factors such enzyme was from, either fungal, bacterial or
as pH, temperature, and ionic strength. All plant.
enzymes are functionally specific to varying Glucose was used as the
degrees. substrate of this
Enzyme activity is the rate of the catalyzed experiment in which the
reaction. When it is plotted against either pH or enzyme acts to. Glucose
temperature, the curve usually has a peak or the 𝐶6 𝐻12 𝑂6 is a
optimum. The region of optimum pH or mosaccacharide sugar
temperature for activity is not necessarily at or which the product of
near the pH of temperature values normal for the photosynthesis in plants. It
living cell from which the enzyme was taken. Not is the source of energy in Figure 2 Chemical
structure of glucose
even the region of optimum pH for activity the cell function from the
necessarily at or near the region of the optimum circulating free sugar in
pH of for stability, which is also the same as to blood and a major participant in metabolism.
temperature. Glucose and fructose make up sucrose. Glucose
units in long chains make to extract invertase from the Baker’s yeast and
up polysaccharides. to determine the effects of changes in pH and
Glucose is commonly used temperature on the reaction rates of an enzyme-
in foods, medicines, catalyzed reaction.
brewing, and wine making
as the source of various EXPERIMENTAL
other organic chemicals. It 1. Glucose Assay Using Saturated Picric
is also known as D- Acid Colorimetric Method
Figure 3 Chemical
glucose, D-glucopyranose, structure of picric A set of test tubes were prepared by following
grape sugar, corn sugar, acid the table below. (See Table 1) All test tubes were
dextrose and cerelose. incubated in 60°C water bath for five (5)
Picric acid 𝐶6 𝐻2 𝑁𝑂2 𝑂𝐻 is a poisonous, minutes. A 1.0 ml saturated picric acid solution
explosive yellow crystalline solid that is and a 1.0 ml 20% sodium carbonate 𝑁𝑎2 𝐶𝑂3 in
commonly used in explosives, dyes, and aqueous solution were added in each of the test
antiseptics. It melts at 122°C and is soluble in tubes then it was diluted in distilled water until it
most organic solvents. It is Picric acid is a reached 10 ml. The test tubes were subjected to
derivative of phenol. It reacts with metals to form spectrophotometer at 540nm molar absorbance.
metal picrates. Picric acid is also known as The absorbance of test tubes #’s 1 to 6 was
2,4,6-Trinitrophenol. presented in a graphical presentation for
In determining the enzyme activity of the analysis.
invertase through glucose, this experiment aims

Table 1 Test tube preparation for Glucose Assay using Saturated Picirc Acid Colorimetric Method.

Test Blank 1 2 3 4 5 6
Tube No.
Glucose 0.0 0.5 1.0 1.5 2.0 2.5 3.0
Standard
Solution
𝟏𝒎𝒈
𝟏𝒎𝒍
Distilled 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Water

2. Effect of pH on Invertase Activity thoroughly. The test tubes were incubated on

A set of four (4) test tubes was prepared with 60°C water bath for five (5) minutes. A 3.0 ml
a 2.90 ml of 0.1M buffer solution with its glucose solution was added and incubated on
appropriate pH in each of the test tubes. (See 60°C water bath for five (5) minutes. A 1.0 ml
Table 2) saturated picric acid solution and a 1.0 ml 20%
Table 2 Test tube preparation for Effect of pH on sodium carbonate 𝑁𝑎2 𝐶𝑂3 in aqueous solution
Invertase Activity
were added in each of the test tubes. The test
Test Tube 1 2 3 4 tubes were diluted in distilled water until it
No. reached 10 ml and these were subjected to
pH 1 3 5 7 spectrophotometer at 540nm molar absorbance.
A graphical representation was prepared for
A 0.10 ml enzyme stock solution was added in analyzation.
the four (4) test tubes and a blank test tube
which is test tube #5, and then it was mix
3. Effect of Temperature on Invertase
Activity
Different temperature of water bath set-ups
was prepared. (See Table 3) A set of four (4)
test tubes containing 3.0 ml glucose solution was
prepared.
Table 3 Test tube preparation for Effect of
Temperature Invertase Activity
Test Tube 1 2 3 4
No.
Temperature 30°C 50°C 60°C 70°C
Each test tube was incubated separately in its
respective water bath set-up for five (5)
minutes. A blank test tube for each of the four
(4) test tubes was prepared. Each of the blank
test tubes containing 0.8 ml invertase and a 2.9
ml buffer solution was prepared. A 3.0 ml dilute
enzyme solution was added to the four (4) test
tubes then incubated for another five (5)
minutes. A 1.0 ml saturated picric acid solution
and a 1.0 ml 20% sodium carbonate 𝑁𝑎2 𝐶𝑂3 in
aqueous solution were added in each of the test
tubes. The test tubes were diluted in distilled
water until it reached 10 ml and these were
subjected to spectrophotometer at 540nm molar
absorbance. A graphical representation was
prepared for analyzation.
Glucose, and other reducing sugars, reacts
with picric acid by oxidoreduction producing a
colored compound that shows a maximal molar
extinction at 530nm. Glucose is reduced into
nitroamino compound, same as to dinitrosalicylic
acid. Oxidoreduction is a reversible chemical
reaction in which one of the reactions is
oxidation and the reverse reaction is reduction.
Since the absorbance at 540 nm is linearly
dependent on the concentration or mass of
glucose the reaction can be used for
quantification of reducing sugars.
In an enzyme assay, in order to be valid, it
must satisfy at least three conditions: (1)
Activity must be proportional to the 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 reageants
were allowed to react in the process to be
subjected in reading its absorbance or molar
extinction.

RESULTS AND DISCUSSION

A. Glucose Assay Using Saturated Picric
Acid Colorimetric Method Figure 4 Simplest Kinetic Equation - Michaelis-Menten
Table 4 Data of Glucose Assay using Saturated Picric Equation
Acid Colorimetric Method
Absorbance was measured through the use of
Test tube Amount of A540 spectrophotometer which measures the amount
no. Acid- of light transmitted through a sample at a given
hydrolyized wavelength. The read absorbance of standard
Surose test tube was subtracted to blank test tube. This
(mg/ml) will give only the absorbance of the invertase,
1 0.05 0.001 without the additional reagents.
2 0.10 0.000 To graph, the x-axis is the concentration of
3 0.15 0.003 acid-hydrolyzed sucrose in milligram over
4 0.20 0.007 milliliter (mg/ml) and the y-axis is the
5 0.25 0.002 absorbance. The graph shows a straight line, the
6 0.30 0.002 standard’s best fit line and the plotted
absorbance of each test tube. Though the experimental technique. The effectivity of the
accumulated data, the plotted absorbance are invertase was uncertain. The assumed optimum
too far in each other and there is no linear trend pH and temperature has the highest value of
in the original process, there is always be one in absorbance which is pH 7 and 70°C,
the finite block of data taken that will represent respectively. One of the possible faults of the
it. The possible faults in glucose assay were (1) test is the inactivity of glucose due to it was not
inactivity of glucose due to it was not freshly freshly prepared. In addition, the certainty of the
prepared; (2) picric acid was not reactive; and reactivity of picric acid was questionable and the
(3) technical deficiencies of the used used spectrophotometer has technical
spectrophotometer. deficiencies in terms of reading the molar
The “best fit” line of the standard shows the extinction.
increasing enzyme activity in increasing
concentration of the standard. This follows the Table 5 Data of Effect of pH on Invertase Activity
principles of the Law of Mass Action. The binding
pH Amount of A540
of the substrate to the enzyme occurs because of
Acid-
the high specific interaction. In some cases,
hydrolyized
when the enzymes are occupied already by the
Surose
substrate, the graph will orient a downward
(mg/ml)
slope indicating that the enzymes cannot bind
1 - 0.072
the excess substrate. The calculated slope-
3 - -0.022
intercept form using Microsoft Excel application
is 𝑦 = 0.0004𝑥 + 0.001. 5 - -0.015
The graphical presentation for the glucose 7 - 0.115
assay using saturated picric acid method is found
in the next page. Table 6 Data of Effect of Temperature on Invertase
B. Effect of pH and Temperature on Activity

Invertase Activity
Temperature Amount of A540
The optimum pH of the invertase is 4.5. The
(°C) Acid-
enzyme is fully active between pH 3.0 to 5.5. A
hydrolyized
pH over 6.0 and above denatures the enzyme. Surose
The optimum temperature of the invertase (mg/ml)
should be at 60°C (140°F). For the invertase to 30 - 0.119
be exposed in elevated temperature and extreme
50 - -0.001
pH after reaching its optimum, the enzyme will
60 - 0.248
slope down indicating denaturation of enzyme. It
70 - 0.485
indicates that an enzyme’s reactivity is controlled
and occurs to a limited extent in the biological
The graphical presentations for the effect of pH
reactions.
and temperature on invertase activity are found
In the experiment, both of the graphs did not
in the next page.
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
 Glucose Assay using Saturated Picric Acid and
20% Sodium Carbonate Method
0.008

0.007

0.006
Absorbance540

0.005

0.004 y = 0.0004x + 0.001

R² = 0.109 Absorbance540
0.003
Linear (Absorbance540)
0.002

0.001

0.000
0.05 0.10 0.15 0.20 0.25 0.30
Amount of Acid-hydrolyized Surose (mg/ml)

Figure 5 Standard "Best Fit" Straight Line of Glucose Assay using Saturated Picric Acid and
20% Sodium Carbonate

Effect of pH on Invertase Activity

0.140
0.120
0.100
0.080
Absorbance540

0.060
0.040
0.020
0.000
-0.020
-0.040
1 3 5 7
Absorbance540 0.072 -0.022 -0.015 0.115
pH

Figure 6 Graph of Effect of pH on Invertase Activity

 Effect of Temperature on Invertase Activity
0.600

0.500

0.400
Absorbance540

0.300

0.200

0.100

0.000

-0.100
30 50 60 70
Absorbance540 - 0.119 -0.001 0.248 0.485
Temperature (°C)

Figure 7 Graph of Effect of Temperature on Invertase Activity

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df
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From online websites and researches: http://www.cababstractsplus.org/abstract

BIERMAN, H.R. & DOAN, F.J. A s/Abstract.aspx?AcNo=20053146793
COLORIMETRIC PICRIC ACID METHOD Accessed on February 1, 2010
FOR DETERMINING LACTOSE
http://jds.fass.org/cgi/reprint/7/4/381.pdf http://www.invertase.net/double.htm
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