Biochemistry Laboratory (BCM 362L), Experiment No.

2, © January 24, 2006

3nd Quarter A.Y. 2005-2006

Subcellular Components

Mr. *****1, *****2, *****2

1
Professor, School of CHE-Chm, Mapua Institute of Technology
2
Student, BCM 362L/ A31, School of CHE-Chm, Mapua Institute of Technology
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ABSTRACT

Cells are the smallest structures capable of components based on its density, mass or size
basic life processes. It contains macromolecules therefore obtaining a supernatant and sediment.
which are proteins, nucleic acids (DNA and Several tests were made to determine how
RNA), carbohydrates and lipids. This experiment positive the homogenized sample is for a
focuses on the separation and identification of particular molecule.
these biomolecules in chicken liver. The
separation was done by sedimentation or Keywords: centrifugation, sedimentation,
generally called as centrifugation. It separates the homogenized, biomolecules, macromolecules
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INTRODUCTION The chicken liver tissue was homogenized by

The components of cells are molecules,
nonliving structures formed by the union of
atoms. Small molecules serve as building blocks
for larger molecules. Proteins, nucleic acids,
carbohydrates, and lipids, which include fats and
oils, are the four major molecules that underlie
cell structure and also participate in cell
functions. For example, a tightly organized
arrangement of lipids, proteins, and protein-
sugar compounds forms the plasma membrane,
or outer boundary, of certain cells. The
organelles, membrane-bound compartments in
cells, are built largely from proteins.
Biochemical reactions in cells are guided by
enzymes, specialized proteins that speed up
chemical reactions. The nucleic acid
deoxyribonucleic acid (DNA) contains the
hereditary information for cells, and another
nucleic acid, ribonucleic acid (RNA), works with
DNA to build the thousands of proteins the cell
needs.1, 2
METHODOLOGY
The solutions used in this experiment were
0.16 M NaCl, 0.1 M HCl, 0.1 M NaOH,
concentrated H2SO4, 1% ribose solution, and 1%
albumin. The reagents used for qualitative
analysis were Biuret reagent, Schiff’s reagent,
Orcinol reagent, and Molisch reagent. The sole
equipment used was a centrifuge.
distilled water in an iced bath. The homogenate
were centrifuged at 2900 rpm for 10 minutes.
The supernatant (supernatant 1) was decanted
into a beaker. 10 mL of distilled water was added
to the sediment (sediment 1) and was mixed to
form a suspension. The supernatant 1 was
centrifuged at 14,700 rpm for 10 minutes. The
resulting supernatant (supernatant 2) was
decanted into a test tube while the sediment
(sediment 2) was treated the same way as in
sediment 1. Sediment 1, sediment 2, and
supernatant 2 is thus ready for analysis.
For analysis of nucleic acid, 2 mL of
sediment 1, sediment 2, and supernatant 2 were
added to a micro test tube along with 2mL of 0.1
M HCl. Marbles were placed on top of the test
tubes, it was heated in a boiling water bath for 20
minutes.
In testing for DNA, diphenylamine test were
performed. 10 drops of hydrolyzed test solutions
were added to the test tubes. 2 drops of
diphenylamine reagent and 1 drop concentrated
H2SO4 were also added. It was then heated in a
water bath until a change in color is observed for
positive indication. “Feulgen’s Nucleal reaction”
test was also performed for determination of
DNA. 10 drops of hydrolyzed test solutions were
added to the test tubes. 0.1 M NaOH were added
drop wise to neutralize the solutions, it was
checked using litmus paper. 2 drops of Schiff’s
reagent were added to each test tube. The test
 tube was then covered with aluminum foil. LITERATURE CITED
Change in color indicates present of DNA.
For the RNA test, 10 drops of hydrolyzed test 1. Biochemistry, Garrett and Grisham, 3rd
solutions were added to the test tubes. 5 drops of Edition
freshly prepared orcinol reagent was added. It
was then heated in a water bath until a change in 2. © 1993-2003 Microsoft Corporation. All
color is observed for positive indication. rights reserved.
A Molisch test was performed to test for
carbohydrates. 5 drops test solutions and 5 drops
Molisch reagent were added into 4 test tubes
(including 1% ribose solution) each. It was
shaked well and tilted at approximately 45º. 1
mL concentrated H2SO4 was added drop wise
down the sides of the test tube. It was slowly
then brought to an upright position. The color
formation was observed.
To test for proteins, a “Biuret test” was
performed. 5 drops test solutions and 5 drops
Biuret reagent were added into 4 test tubes
(including 1% albumin solution). It was shaked
well and positive indication was observed.

RESULTS AND DISCUSSION

In this experiment, distilled water was used as

homogenizing medium which is capable of
breaking up the cell membrane. Several tests
were performed for the identification of the
subcellular component and the results are shown
below:

Test Control Sed 1 Sed 2 Sup 2

Carbohydrate +++ + + +
Protein +++ +++ + +++
RNA N/A +++ + +++
D Diphenylamine Test N/A ++ + ++
N
Feulgen’s Test N/A + ++ N/A
A

From the above table one can conclude which

biomolecule is abundant in the cell, in this case
RNA and Protein are.
The experiment is limited to two sediments
and supernatants because of limited capabilities
of the centrifuge. Note that sample is kept at low
temperature most of time to preserve the cells.

CONCLUSION

Bigger or heavier molecules are found in the

sediment, while the lighter on the supernatant
liquid. Homogenizing liquid can greatly affect
the qualitative analysis which depends on the
breakdown of cellular components.