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Thiara Terri V. Bella, Terrence Louis P. Carlos, Joseph Bernard E. Cordova, Jose Alfonso P. Cuisia and
Crystel Mhariel V. Daroy
Group 2 2F Medical Technology General Biohemistry Laboratory
ABSTRACT
Enzymes are biological molecules(proteins) that act as a catalyst and help complex reactions occur everywhere in life.
Invertase is an enzyme that catalyzes the breakdown of sucrose to form glucose and fructose. Invertase was
extracted from Bakers yeast and the substrate was collected to determine the effects of changes in pH on the reaction
rates of an enzyme-catalyzed reaction. 2.90 ml of buffer was placed in 7 test tubes having a pH of 2,3,4,5,7,9, and
11. The enzyme stock solution was poured and incubated. At the end of the experiment, spectrophotometry was used
at 540nm to be able to measure its absorbance.
INTRODUCTION
Enzymes are complex proteins that cause a
specific chemical change in all parts of the body.
For example, they can help break down the foods
we eat so the body can use them. Blood clotting
is another example of enzymes at work.
sugars and other reducing molecules to form 3amino-5-nitrosalicylic acid, which absorbs light
strongly at 540 nm[3] thus the need to use
spectrophotometry to measure the solutions
absorbance.
EXPERIMENTAL
A. Compounds tested
For extraction of invertase from yeast:
0.25 g of Bakers yeast and 150mL
distilled water.
For preparation of denatured invertase
stock solution: 100mL enzyme stock
solution, and boiling water bath.
2. Preparation
of
Denatured
Invertase Stock Solution
100mL enzyme stock solution was incubated in
a boiling water bath for 10 minutes. The solution
was allowed to cool and the supernatant can only
be collected if frothing or formation of
overflowing mass of bubbles occurs. This serves
as the denatured enzyme stock solution that will
be used for the succeeding experiments.
3. Effect of pH on Invertase Activity
7 numbered test tubes was prepared and 1 test
tube for the blank solution. 2.90mL of buffer was
poured on every test tube with the pH 2, 3, 4, 5,
7, 9, and 11. 0.10 mL enzyme stock solution was
added to each solution, mixed thoroughly,
incubated for 60 degrees Celsius water bath for 5
minutes. 1.50mL of sucrose solution was added
and incubated again for 5 minutes in a 60
degrees Celsius water bath. 3mL of DNS reagent
was added then immersed in a 95 degrees
Celsius water bath for 5 minutes to develop the
characteristic red-brown color. The solutions was
allowed to cool. Blank solution was prepared by
following the steps mentioned but the denatured
enzyme was used instead of the enzyme stock
solution. Absorbance was measured at 540nm.
The amount of sucrose hydrolyzed was
determined using hydrolyzed-sucrose standard
curve
constructed
in
the
dinitrosalicylic
colorimetric method.
Absorbance at 540nm
-0.004A
-0.012A
-0.011A
5
7
9
11
0.011A
-0.008A
0.004A
-0.002A
REFERENCE:
[1] Retrieved from http://www.worthingtonbiochem.com/introbiochem/effectsph.html
on Mar. 17, 2015
[2] Retrived from
http://cdn.intechopen.com/pdfswm/26595.pdf on Mar. 17, 2015