THE BIOMOLECULES IN SOME CELL ORGANELLES

Abiog M.L., Arcellana J.E., Pangilinan C.J.

INTRODUCTION
Cell organelles have biomolecules such as Nucleus, Mitochondrion, Lysosome
and Endoplasmic reticulum. Nucleus contains the DNA and its chromosomes and
serves as the cells control center. DNA replicates itself and its daughter cells become
replicates of the parent DNA. The Mitochondrion serves as the powerhouse of the cell.
It supplies power to the activities of the cell. Mitochondria break down small carbon
containing CO2 ad H2O and in this process it releases energy that is stored in ATP which
diffuses throughout the cell and gives fuel to the cells biochemical transaction of life.
Lysosome is a small, sphere-shaped vesicle that can be found in eukaryotic cell. It
contains enzymes that are powerful and have the capability of breaking down worn-out
organelles and ship to the cytoplasm their building blocks where it can be reused to
create new organelles. Lysosomes also recycle proteins, lipids and other molecules.
Lastly, the endoplasmic reticulum, an elongated membranous sac attached to the
nuclear membrane that serves as the tunnels through the cytoplasm. It has two forms,
the smooth endoplasmic reticulum which synthesizes lipids, phospholipids, and steroids
(Nakasako M, Nomura H, Ogawa H 2000)and rough endoplasmic reticulum which
serves as the site for protein synthesis (Nelson D.L, Cox M. 2013).

One of the most known experimental approaches to know the individual reaction
is the study of cell tissue dispersion which includes the breaking of the cells membrane
and the releasing of the cells content (Nelson D.L, Cox M. 2013). Dispersion can be
done by centrifugation, a process which involves the application of the centripetal force
for the sedimentation of heterogeneous mixtures with a centrifuge (Garrett, Reginald H.;
Grisham, Charles M. 2013). In this scientific paper, the principle involved is the lysis or
disruption of cell membrane by mild homogenation, a process whereby a biological
sample is brought to a state such that all fractions of the sample are equal in
composition (Chang, Ta-Yuan et al. 1981), and is carried in isotonic sucrose solution.
The organelles such as nuclei, mitochondria, lysosomes and ribosomes remain intact.
They can be isolated through differential centrifugation. The components can be
precipitated through centrifugation at different rate. The fractions may be tested in vitro
to know the presence of biomolecules. (Nelson D.L, Cox M. 2013)

This scientific paper aims to isolate the organelles of the cell and make a list of
the organelles that were separated through centrifugation. This paper also aims to
indicate the different biomolecules that are present in each of the organelles and apply
test that will distinguish the biomolecules present in selected organelles.

MATERIALS AND METHODS

10 grams of freshly excised blood-free chicken liver was washed with 0.25 M
sucrose solution and dried with filter paper afterwards gets its weight. 50 ml of 0.25 M
sucrose solution was placed in a mortar together with the liver and grinded finely then
placed in a centrifuge tubes.
The mixture was homogenized by means of differential centrifugation
A. The mixture was centrifuged at 600 x g for 10 minutes, transferred and labeled the
supernatant liquid as filtrate I. 10 ml of 0.25 sucrose solution was added to the gathered
residue, and labeled as precipitate I. The suspension contains nuclei and unbroken cells
and tested for the presence of biomolecules.
B. Filtrate I centrifuged at 15,000 x g for 20 minutes transferred and labeled as filtrate II.
10 ml of 0.25 sucrose solution was added to precipitate II to form a suspension.
Mitochondria were present in this suspension and tested for the presence of
biomolecules.
C. Filtrate II centrifuged at 20,000 x g for 30 minutes, poured and labeled as filtrate III.
The gathered residues were mixed with 10 ml of 0.25 sucrose solution. The suspension
comprises lysosome and tested for presence of biomolecules.

Tests for biomolecules.

A. Protein. Biuret tests

5 drops of the suspension were mixed with 5 drops of 10% sodium hydroxide
solution in test tube and 5 drops of 0.5% of copper sulfate solution were added. The
violet-pink color produced by the solution was observed.

B. Carbohydrates. Molisch test

10 drops of the suspension were mixed thoroughly with 4 drops of Molisch
reagent in a test tube. 10 drops of concentrated sulfuric acid were carefully added to the
side of the tube without shaking it. Purple color formed the solution was taken note.

C. Lipids. Salwoski test

Caution: This procedure was performed away from the Bunsen burner since the ether is
highly flammable.

8ml of the solution was placed in the evaporating dish with the use of pipette,
heated it to dryness and cooled. 2 ml of ether was added to extract the residue,
repeated 3 three times. All the ether extract were combined in a test tube and allowed to
evaporate then 2 ml of chloroform were added again.
Caution: Do not inhale chloroform since it is toxic, and avoid contact of this chemical
with your skin or eyes.
10 drops of the chloroform solution were carefully mixed with 5 drops of
concentrated sulfuric acid in a test tube. The color of the sulfuric acid layer, which is
yellow with green fluorescence while the chloroform layer, which is bluish red gradually
turning to violet-red was observed and recorded.

D. Nucleic acids
1. Isolation of nucleic acids
1M NaCl solution was used to saturate 2ml of the suspension, strained using
filter paper then the precipitate was discarded. To precipitate the nucleic acids, ethyl
alcohol was added to the filtrate. (This was used as cell suspension.)
a. DNA: Disch test
5 drops of cell suspension and 5 drops of diphenylamine were added in a
test tube. 6 drops of concentrated sulfuric acid was slowly added to it. The blue
color formed was observed. It was heated in a boiling water bath for 5 minutes.
The test was observed and recorded.
b. RNA. Orcinol test
5 drops of freshly prepared Orcinol reagent was added to 5 drops of the
cell suspension. It was heated in a boiling water bath for 3 minutes. The test was
observed and recorded.

RESULTS AND DISCUSSION

Biomolecules
Tested For
A. Protein. Biuret
Test

Precipitate 1

Precipitate 2

Precipitate 3

Positive
The solution turns
from blue to violet.
(Proteins are
present)

Positive
The solution turns
from blue to violet.
(Proteins are
present)

Negative
The solution turns
blue (Proteins are
absent)

Negative
Brownish-orange
centrifugate formed
instead of the
appearance of a
purple ring at the
interface between
the acid and test
layers
(Carbohydrates are
not present)

Negative
Brownish-orange
centrifugate formed
instead of the
appearance of a
purple ring at the
interface between
the acid and test
layers
(Carbohydrates are
not present)

Positive
The solution turns
into reddish-purple.
(Carbohydrates are
present)

B. Carbohydrates.
Molisch test

C. Lipids. Salkowski
test

Negative
Clear centrifuged
formed instead of
red. (Lipids are not
present)

Positive
Negative
The reagent turns to Clear centrifuged
brick red color
formed instead of
(Lipids are present) red. (Lipids are not
present)

Positive
Blue color indicates
the presence of
DNA

Positive
Blue color indicates
the presence of
DNA

Positive
Blue color indicates
the presence of
DNA

Positive
Yellow color
indicates the
presence of RNA

Positive
Yellow color
indicates the
presence of RNA

Positive
Yellow color
indicates the
presence of RNA

D. Nucleic Acids
(DNA test)
(RNA test)

1. What is the significance of differential centrifugation?

Differential centrifugation is a common procedure in microbiology and cytology
used to separate certain organelles from whole cells for further analysis of specific parts
of the cells. It is also the simplest form of separation, sometimes called differential

pelleting particles of different densities or sizes in a suspension will sediment at different

rates, with the larger and denser particles sediment faster. These sedimentation rates
can be increased by using centrifugal force. A suspension of cells subjected to a series
of increasing centrifugal force cycles will yield a series of pellets containing cells of
decreasing sedimentation rate.

Particles of different densities or size will sediment at different rates with the
largest and most dense particles sedimenting the fastest followed by less dense and
smaller particles.

Differential pelleting is commonly used for harvesting cells or producing crude

subcellular fractions from tissue homogenate. For example, a rat liver homogenate
containing nuclei, mitochondria, lysosomes, and membrane vesicles that is centrifuged
at low speed for a short time will pellet mainly the larger and more dense nuclei.
Subsequent centrifugation at a higher centrifugal force will pellet particles of the next
lower order of size (mitochondria) and so on. It is unusual to use more than four
differential centrifugation cycles for a normal tissue homogenate.

Due to the heterogeneity in biological particles, differential centrifugation suffers

from contamination and poor recoveries. Contamination by different particle types can
be addressed by resuspension and repeating the centrifugation steps (washing the
pellet)

2. Enumerate the different cell organelles and give their composition and functions.
Mitochondria - produces energy through cellular respiration
Rough endoplasmic reticulum - transport and storage
Ribosomes - create proteins
Smooth endoplasmic reticulum - creates lipids or fat
Chloroplast - creates glucose
Golgi apparatus - synthesis, packages and releases concentrate proteins or lipids
Golgi body - protein or lipid enters the cytoplasm
Cytoplasm - where all chemicals take place
Glycoprotein - short sugar chains attached to proteins
glycol lipids - lipids attached to proteins
Cisternae - flattened stacked membrane folds

Liposome - small membrane bounded transport vesicles

Peroxisome - micro bodies found in animal cells
Glyoxysome - micro bodies found in plant cells
Centrioles - for cellular division and cellular reproduction
Cytoskeleton - supports structure and helps move synthesized proteins
Lysosomes - contain hydrolytic enzymes for digestion
Cilia - hair like structures
Flagellum tail contractile
Vesicle - moves protein, lipid and carbohydrate
Nuclear envelope - surrounds the nucleus
Vacuole - contains food or water
Cell membrane - separates cell contents from the environment
Microtubules - provide internal support
Nucleus - information center of the cell
Nucleolus - site of ribosome synthesis
Chromatin - threadlike mass of DNA

3. How is 500mL of 0.25-M sucrose solution prepared?

Molar mass sucrose C12H22O11 = 342.3g/mol
0.25mol = 342.3*0.25 = 85.58g
In 0.5L = 85.58/2 = 42.79g
Weigh out 42.79g and dissolve in water. Make up final volume to 500mL.
For 1 M Sucrose solution, 342g sucrose will be added in distilled water to make up the
final volume to 1000 ml. therefore, for 0.25 ml, exactly one fourth, 85.5 g of sucrose will
be added in distilled water to make up the final volume to 1000ml. This will result in the
concentration of 0.25 M.
4. List the biomolecules present in each of organelles that are separated in the process
of fractional centrifugation.
A. nucleic acids
B. proteins
C. lipids
D. carbohydrates mainly glycogen which are mostly found in the liver

SUMMARY

Different test were performed to confirm the presence of biochemical molecules.

Same tests were performed to the 3 filtrates who undergone differential centrifugation.
There are times where differential centrifugation was performed and the results were
different in the same test. But sometimes, the result is unchanged. Like the nucleic acid
test where the results werent changed from filtrate 1 to filtrate 3. Both the DNA test and
the RNA test yield to a positive result for the 3 filtrates. DNA test were done by
combining the suspension and diphenylamine. Positive results will yield to a blue color
which then shows that the 3 filtrates were positive. RNA test were done by adding
orcinol which yield to a yellow color as a positive results as seen in the table. But unlike
to the nucleic acid test, the tests for lipids, carbohydrates and protein does not yield
consistent result. For protein test, the 3rd precipitate shows a negative result or
formation of blue color. Positive result will yield to blue to violet color in the addition of
10% Sodium hydroxide. And for the test of carbohydrates, addition of molisch reagent
shows presence of carbohydrates on the 3rd precipitate which shows a redish-purple
color. Lastly, for the test for lipids which yield a positive result on the 2 nd precipitate when
salwoski test were performed, this shows a brick red color.

REFERENCES

J. Graham, Biological Centrifugation, p. 5.

J. Graham, Biological Centrifugation,p. 24.
D. Rickwood, J.M. Graham (2001). Biological Centrifugation. Springer Verlag; ISBN:
0387915761

Nelson D.L, Cox M. (2013) Leningher, Principles of biochemistry (6 th ed)

Nakasako M, Nomura H, Ogawa H (2000)