QUALITATIVE TESTS HYDROLYSATE UMHYDROLYZED RNA

Benedict’s Test for Violet No change of color remained

Reducing Sugars blue
Orcinol Test for Pentoses Green blue solution light blue solution
Test for Purine Bases White ppt Small white ppt
Test for Inorganic Yellow ppt Yellow ppt
Phosphate

HYDROLYSIS OF NUCLEIC ACIDS

A. ACID HYDROLYSIS OF RNA

Acid hydrolysis is a process in which a protic acid is used to catalyze the cleavage of a chemical
bond via a nucleophilic substitution reaction, with the addition of the elements of water (H2O).
RNA acid hydrolysis occurs when the deprotonated OH of the ribose acting as a nucleophile, reacts
with the adjacent phosphorus in the phosphodiester bond of the sugar-phosphate backbone of the
RNA. Due to the extra OH group that the ribose backbone has, which allows it to be better
hydrolyzed in alkaline conditions rather than acid. Acid hydrolysis cleaves susceptible purine N-
glycosyl bonds in both DNA and RNA but when RNA is boiled in dilute acid, adenine and guanine
are released, leaving an “apurinic acid” which may be further hydrolyzed to a mixture of
pyrimidine nucleotides. Bond cleavage of the N-glycosyl bonds requires more vigorous conditions
like heating along with adding acid. This would release cytosine and uracil. However, during the
process, there is a tendency for cytosine to be deaminated to uracil.

In the experiment, the acid hydrolysis of RNA is obtained by treatment with 10% H2SO4 heated
in boiling water bath for about 1 hour. Complete hydrolysis of RNA yields a pentose sugar,
phosphoric acid, and nitrogen containing heterocyclic compounds.
 B. QUALITATIVE TESTS

Test for Reducing Sugars

Benedict’s Test was used in the experiment to determine presence of sugars in both
hydrolyzed and unhydrolyzed RNA. When reducing sugars are mixed with Benedicts reagent and
heated, a reduction reaction causes the Benedicts reagent to change color. This reaction is caused
by the reducing property of simple carbohydrates. The copper (II) ions in the Benedict’s solution
are reduced to Copper (I) ions, which causes the color change. RNA is a nucleotide containing
reducing sugars, during hydrolysis RNA releases ribose sugar, sugar base and phosphate, enabling
it to react with Benedict’s solution producing a brick-red precipitate.

Theoretically the hydrolyzed sample produces a brick-red colored precipitate, in the

experiment the hydrolyzed sample yielded a violet solution, indicating an error in the performed
experiment. The unhydrolyzed sample produced a light blue solution indicating a negative result
for the presence of reducing sugar in unhydrolyzed RNA.

Ribonic Acid

Ribose Sugar

Test for Pentoses

Orcinol test is a test for the determination of the presence of Pentoses in a hydrolyzed and
unhydrolyzed RNA sample. The test occurs when the pentose in ribose converts in the presence of
hot acid to form furfural, which reacts with Orcinol reagent (3,5 dihydrotoluene) in the presence
of ferric chloride present in the Orcinol reagent and catalyst purine to form a blue-green colored
product. For the unhydrolyzed sample of RNA concentrated HCl is used to dehydrate the ribose,
a pentose sugar present in the RNA molecule, into a furfural. In the experiment the presence of
blue-green solutions indicated positive for pentoses present in the samples.
Test for Purine Bases

Purine Bases are known as the two-carbon nitrogen ring bases, adenine and guanine both
of which are present in RNA. Test for purine bases gives the reaction of RNA with nitric acid
given that purines are known to be readily soluble in dilute acids. In the experiment we used
ammonium hydroxide and silver nitrate, which produced a flocculent, gelatinous white precipitate.
The flocculation is due to the suspension of particles that coalesce, or flocculate during the
sedimentation process. Based on the experiment the hydrolyzed RNA gave clearer results and
presence of white precipitate in comparison to the unhydrolyzed RNA.

Test for Inorganic Phosphate

The qualitative test is used to determine the presence of Inorganic phosphate in hydrolyzed
and unhydrolyzed RNA sample with ammonium molybdate solution. The test hydrolyzes
pyrophosphate to phosphate and reacts with the reagent, ammonium molybdate to produce
ammonium molybdophosphate to produce a yellow precipitate. In the experiment both solution
produced yellow precipitate indicating positive for the presence of Inorganic phosphate in
hydrolyzed and unhydrolyzed RNA.

HPO42-(aq) + 12MoO42-(aq) + 3NH4+ (aq) + 23H3O+ (aq) (NH4)3 [P(Mo3O10)4] yellow

Conclusion

RNA can be extracted from yeast by rupturing yeast cells through increasing its PH level
to denature unwanted and contaminant proteins, lower the PH level to denature contaminant
proteins and substances, and centrifuge is done to separate denatured substances and other
unwanted substance to isolate yeast.

Different tests were performed in order to identify the content and composition of RNA.
Acid hydrolysis done in the RNA sample causes depurination, cleaves the purine N- glycosyl
bonds, liberating adenine and guanine and apurinic site remains. Some phosphodiester bond are
cleaved, much harsher and acidic is required to remove pyrimidine N-glycosides. In the
experiment the hydrolyzed RNA indicated the presence of pentoses, purine bases, and inorganic
phosphate by showing positive results in the test theoretically hydrolyzed RNA is also positive for
Benedict’s test but is shown otherwise in the performed experiment due to some errors. While
unhydrolyzed RNA showed positive for test for pentoses and inorganic phosphate only due to
unliberated components of the RNA in the unhydrolyzed sample.
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