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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


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