Ribonucleic acid (RNA) Chemical Characterization

Moron, R.S.S., Pazon, A.D., Ramirez, C.V., Raquepo, T.M.R., and Razon, D.N.A, Jr. Recabo,
P.P.L.
2B-Pharmacy, Group No. 6, Department of Pharmacy, Faculty of Pharmacy,
University of Santo Tomas, Espaa Boulevard, 1015 Manila, Philippines
ABSTRACT
The purpose of the experiment is to determine the behavior of Ribonucleic Acid (RNA) towards
various qualitative color reaction tests following alkaline hydrolysis. During Alkaline hydrolysis,
A portion of the isolated RNA was subjected using 2mL 0.3 M NaOH. The RNA hydrolysate was
characterized by different tests: Test for Ribose, Test for Phosphate, Test for Purines (Murexide
Test) and Test for Pyrmidines (Wheeler-Johnson Test). For the test for ribose, the hydrolyzed
RNA yielded a light green solution while the standard ribose solution yielded a dark green
solution. For the test for phosphate the hydrolyzed, unhydrolyzed and standard phosphate
solution yielded clear solutions with no precipitate. For the test for purines, the hydrolyzed,
unhydrolyzed and standard guanine all yielded yellowish-orange residues. For the test for
pyrimidines, both the hydrolyzed and unhydrolyzed RNA both yielded transparent liquids with
white precipitate while the standard cytosine yielded a purple solution.
INTRODUCTION
Nucleic acids are biomolecules important
for their roles in the storage, transfer and
expression of genetic information. Two
fundamental types of nucleic acids
participate
as
genetic
molecules:
deoxyribonucleic
acid
(DNA)
and
ribonucleic acid (RNA). DNA is found
primarily in the chromosomal form in the
cells nucleus, where it serves as the
repository of genetic information. [1]
On the other hand, RNA has a wider range
of functions which includes protein
synthesis. RNA is a biologically important
type of molecule that consists of a long
chain of nucleotide units. If DNA is usually
double stranded, RNA is basically a single
stranded nucleic acid. [4] It is usually found
at high concentration in large cytoplasmic
volume due to its specific functions. It is

present in three major types: ribosomal RNA

(rRNA), messenger RNA (mRNA) and
transfer RNA (tRNA). Each of these three
forms plays a role in the expression of the
genetic information in DNA. Messenger
RNA carries the transient message for
protein synthesis from nuclear DNA to the
ribosomes. Transfer RNA, the smallest
nucleic acids, form esters with specific
amino aicds for use in protein synthesis. It
serves as adapter molecules for the
translation of information in mRNA into a
specific sequence of amino acids. Ribosomal
RNA, the most abundant form, is associated
with protein-synthesizing organelles, the
ribosomes. [5] Other classes of RNA include
small nuclear RNA (snRNA), micro RNA
(miRNA) and small interfering RNA
(siRNA) which are also important in
affecting gene expressions.[6]

Nucleic acids are linear polymers

constructed from four different monomers
called nucleotides. Nucleotides have three
characteristic components: a nitrogenous
base, a pentose sugar and a phosphate.[2] All
nitrogenous bases in DNA and RNA are
derivatives of the two heterocyclic
compounds purine and pyrimidine. The
major purines in DNA are adenine and
guanine; the major pyrimidines in DNA are
thymine and cytosine. Similarly, the
predominant purines in RNA are adenine
and guanine; however, the pyrimidines in
RNA are cytosine and uracil. Two types of
aldopentoses are found in nucleic acids.
Ribose occurs in RNA; 2-deoxyribose in
DNA. [3] DNA lacks a hydroxyl group
attached to the pentose ring in the 2 position
which makes RNA less stable than DNA
because RNA is more prone to hydrolysis. [3]
METHODOLOGY
Reagents used
The materials and reagents used were .3M
NaOH, 10% KOH, Conc. H2SO4, Conc.
HNO3, Ba(OH)2, 10% (NH4)2MoO4 solution,
Br2 water, Orcinol reagent and the standard
solutions namely :ribose, adenine, guanine,
cytosine and uracil.
Procedure
1. Alkaline Hydrolysis
The mixture solution of 2 ml of 0.3N
NaOH and a small amount of isolated RNA
was heated via water bath for 60 minutes.
The hydrolysate was cooled and adjusted to
pH 4-6 with glacial acetic acid using pH
paper. The hydrolysed sample RNA was
used for testing.
2. Test for Ribose

A mixture of 0.5 ml hydrolyzed RNA

solution and 2 ml Orcinol reagent was
placed in a water bath for 5-10 minutes. The
same method was used for the standard
ribose solution.
3. Test for Phosphate
1ml of conc. H2SO4 was mixed with the
nucleic acid solutions (e.g. hydrolysed,
unhydrolyzed and standard phosphate). The
tubes were then heated over a small flame
with constant shaking until it went brown.
After cooling, .5ml HNO3 was added and
was heated again until white fumes
appeared. 1ml water was added to the liquid
and was heated for 5 minutes in a boiling
water bath and was then cooled and was
again added 1mL 10% % (NH4)2MoO4
solution. After they were mixed the tubes are
mixed and diluted to 10 mL using water.
After letting it stand for 5 minutes the color
and precipitate was observed.
4. Test for Purines (Murexide Test)
5-10 drops of RNA solution (e.g.
hydrolysed, unhydrolyzed and standard
guanine) was placed in a small evaporating
dish. Few drops of concentrated HNO3 were
added to the solution and evaporated to
dryness on a hot plate. The residue formed
was moistened with 10% KOH and heated
to dryness. Few drops of water were added
to the dried solution. Same procedure was
done to the standard guanine solution and
unhydrolyzed RNA solutions. Any change in
color was noted.
5. Test for Pyrimidines (WheelerJohnson Test)

An excess of bromide water was added

to 0.5 mL RNA solution until the solution
turned yellow. The solution was boiled on a
hot plate until a change in color to light
yellow or colorless occurred. An excess of
Ba(OH)2 was added to the solution and
tested with litmus paper. Same methods
apply for the unhydrolyzed RNA and
standard cytosine. Any change in color was
noted.

III. RESULTS AND DISCUSSION

Table 1. Qualitative Colour Reaction Tests
results of Standard RNA solution and RNA
Hydrolysate and unhydrolyzed RNA.
Test
Ribose

Hydrolyzed Unhydroly Standard

RNA
zed RNA
solutions

Light
Dark Green
Green soln.
soln.
Phosphate
Clear sol, Clear sol, Clear sol, no
no ppt.
no ppt.
ppt.
Purines
Yellowish- Yellowish- Yellowishorange
orange
orange
residue
residue
residue
Pyrimidine Clear sol, Clear sol, Clear
sol,
s
white ppt.
white ppt. white ppt.
1. Test for Ribose
Positive results for the standard solution
(dark green solution) and for the hydrolysed
RNA from yeast ( light green solution) were
yielded due to complete conversion of the
ribose to an aromatic aldehyde (furfural)
which when reacted with Orcinol reagent
(3,5-dihydroxy
toluene)
formed
an
aldehyde-phenol condensation.

Figure 1. Reaction of ribose with orcinol

reagent .
2. Test for Phosphate
In the test for the presence of phosphate
in both standard phosphate solution and
RNA hydrolysate, a yellow precipitate
should be ideally obtained however the
group obtained no ppt. The formation of ppt.
is due to the reaction of Ammonium
Molybdate solution which when dropped
upon a sample, indicates the presence of
phosphate by a yellow stain or a crust of
yellow phospho-ammonium molybdate.
Formation of yellow crystals follows.
However, In this test the group did not
achieve a precipitate at the end.
3. Test for Purines (Murexide Test)
In the test for purines, or commonly
known as murexide test, the RNA (both
hydrolyzed and unhydrolyzed) from yeast
and the standard solution yielded a
yellowish-orange residue when oxidized
with nitric acid and evaporated due to purine
degradation. They turned into yellow
residues when moistened with a base, which
is a positive result for presence of purine
bases and turned back to yellowish orange
ppt (standard solution) and yellowish orange
ppt (both hydrolyzed and unhydrolyzed
RNA from yeast) when water was added and
evaporated.

Figure 2. Murexide Test

4. Test for Pyrimidines(WheelerJohnson Test)

In the test for pyrimidines, all of the samples
yielded a clear solution with white
precipitate. Ideally, if the sample is treated
with bromine water it should form 5-bromo-

6-hydroxyhydro derivatives which produces

a yellow coloration. Upon dehydration in
solution, it forms a 5-bromo derivative. The
addition of barium hydroxide Ba(OH)2 gives
a 5, 5-dibromo-6-hydroxyhydro derivatives,
a violet precipitate, which is a positive result
for the presence of uracil in RNA. Both the
standard solution and RNA sample (both
hydrolysed and unhydrolysed did not yield
5, 5-dibromo-6-hydroxyhydro derivative
needed to form a violet precipitate when
treated with Ba(OH)2.
A number of samples gave erroneous results.
There is a possibility that contamination
could have occurred in the course
experiment or mainly due to the poor
purification method employed.

REFERENCES
[1] Nelson, D.L. and Cox, M.M. (2000). Lehninger Principles of Biochemistry, 3rd Edition. New
York: Worth Publishers, pp. 325-328, 345-346.
[2]
Characterization
of
Nucleic
Acids.
Retrieved
from
http://www.docshare.com/doc/83332/Characterization-of-Nucleic-Acids 7:17 PM February 8,
2011
[3] Murray, R.K. (1988). Harpers Biochemistry, 21st ed. Connecticut: Appleton & Lange, pp.
383-386.
[4] Selective Advantage of DNA over RNA as the Genetic Material. Retrieved from
http://www.ncbi.nlm.nih.gov/books/NBK22508/ 8:43 PM February 10, 2011
[5]
RNA.
Retrieved
http://virtualology.com/virtualsciencecenter.com/halloforganicchemistry/RNARibonucleicacid.com/ 6:22 PM February 8, 2011

from

[6] Meili, M., B. Albert-Fournier, and M. C. Maurel. "Recent Findings in the Modern RNA
World." International Microbiology 4 (2001): 5-11.