Experiment 3: Enzymes

Crystal Gale M. Resurreccion, Crizzia Belle B. Reyes, Markus Gerard O. Reyes,

Catherine Joy B. Rodriguez, Ann S. San Juan
Group 7 2E Medical Technology Biochemistry Laboratory

ABSTRACT
Enzymes are compounds responsible for catalyzing chemical reactions in the body.
Enzymatic activity is affected by several factors such as pH and temperature. In this
experiment, invertase was extracted from yeast and was used to undergo several chemical
tests to observe the effects of these factors to its activity.

INTRODUCTION curves which serves as the function of

Enzymes are biological pH, just like the shown example below.
molecules, typically proteins, that
significantly catalyze or speed up the
rate of chemical reactions, usually
within a cell. They reduce the activation
energy which is essential for starting
any type of chemical reaction. The
reaction takes place faster for the
energy requirement for activation is
lower with the aid of the enzymes. An
overall performance of an enzyme
depends on various factors affecting it
Figure 1. Effect of pH on Enzymatic
such as the temperature, pH, its
Activity
cofactors, inhibitors and activators.
Enzymes are classified as
The bell-shaped curve indicates
proteins which are very sensitive to
the stability of the enzymes during the
changes in the pH. For each and every
alteration of pH. This means that when
enzyme, it has its own optimum range
the pH is low, this will result to a slower
for pH which determines where it will
reaction rate. But when a pH is high,
be most active. This results when the
the reaction still remains the same
pH are affected by the following
because of the enzymatic loss. The
factors: (1) the catalytic activity of the
peak in the “bell” or in the graph
enzyme, (2) the binding activity
indicates and demonstrates the “best”
between the enzyme and the
pH,also known as the optimum pH,
substrate, (3) the ionization of the
which is suitable for the enzyme when
substrate and lastly (4) the variation of
it reaches its maximum rate of the
the protein structure. Various
enzymatic activity.
enzymatic reactions and their initial
Dinitrosalicylic acid, also known
reaction rates showcase bell-shaped
as DNSA or 3:5-dinitrosalicylic acid, is
a monohydroxybenzoic acid consisting
of 2-hydroxybenzoic acid having nitro
substituents at the 3- and 5-positions.
It is a yellow colored reagent used in
colorimetric testing for the presence of
free carbonyl groups (C=O) in reducing
sugars. It is also used in determining
sugar content especially glucose. The
DNSA method or technique is utilized
to estimate the quantification of sugar
in various bodily fluids such as blood.
This is also used effectively in handling
the requirements for diabetic clinics in
various hospital laboratories. It is with
consideration that it only takes 10
minutes to process and the said
reagent is known to be stable, cheap
and very easy to prepare.
There are metabolic implications
in quantifying the amount
of sugar in blood and this is used in
determining the presence of blood
sugar-related disorders such as
diabetic hypoglycemia and polydipsia.
The said method, DNSA colorimetric
method is one good way to assess
blood sugar levels that will surely help
a lot of individuals.
EXPERIMENTAL
Extraction of Invertase from Yeast
After weighing ten grams of the
baker’s yeast it was dissolved in 30
milliliters of 0.1M NaHCO₃. The solution
was left in a room temperature for a
day before the supernatant was
decanted. The supernatant was diluted
using distilled water in a 1:100 ratio
which will then be used as the enzyme
solution in the next experiments.
Preparation of Denatured
Invertase Stock Solution
The 25 mL of the enzyme stock
solution was cooled after it was put in
hot water bath for ten minutes. Then,
the supernatant formed in the solution
was collected as denatured enzyme
stock solution for the next experiments
performed.
Effect of Temperature on Invertase
Activity
3 water baths of varying
temperatures (20°C, 50°C, and 70°C)
were prepared, together with 3 test
tubes containing 1 mL of 0.03M
sucrose, 1.4mL of distilled water, and
0.50 mL of 0.05M acetate buffer
solution with a pH of 4.7, each. They
were immersed, separately, in each of
the water baths for 5 minutes. 1mL
enzyme solution was then added to
each of the test tubes and they were
incubated for another 5 minutes. 2mL
of DNS reagent was mixed and the test
tubes were placed in a 95°C water bath
for 10 minutes until a characteristic
red-brown color appears. The test
tubes were then allowed to cool in an
ice water bath and 5mL of distilled
water was added. For the reagent
blank, 1 test tube was prepared,
containing 1 mL of the denatured
enzyme solution, 1 mL of 0.03M
sucrose, 1.4mL of distilled water, and
0.50 mL of 0.05M acetate buffer
solution with a pH of 4.7, and was
incubated for 5 minutes at room
temperature. 2mL of DNS reagent was
put, the test tube was immersed in a
95°C water bath for 10 minutes, left to
cool in an ice water bath, and 5mL of
distilled water was added to the
solution. The test tubes were then
placed in a spectrophotometer to the amount of invert sugar produced in
measure the absorbance at 540 nm each test tube
and the results were plotted against

RESULTS AND DISCUSSION

Table 3. Results on the Effect of

Temperature on Invertase Activity

Tempera Amoun Absorbance5

ture (°C ) t Invert 40
Sugar
Produc Actual Due to time constraints and lack
(AA -
ed of equipment, only 20°C, 50°C, and
(AA) A B)
(mol/L) 70°C water baths were prepared.
However, the effect of temperature on
Reagent 0.000 0.247 0.000 invertase activity can still be
Blank determined from the experiment. The
optimum temperature of the graph falls
20°C 0.070 0.946 0.699 at 50°C. This means that there was an
increase in enzymatic activity during
50°C 0.159 2.067 1.820 this temperature. Above the optimum
temperature, the protein starts to
70°C 0.121 1.589 1.342
denature which decreases the amount
of enzymatic activity. This was clearly
demonstrated as the graph fell during
70°C.

REFERENCE

Crisostomo A. C., Daya, M. L., et al

(2010) Laboratory Manual in General
Biochemistry Quezon City: C&E
Publishing, Inc.