CHEMICAL CHARACTERIZATION OF RIBONUCLEIC ACID

Ma. Angelica C. Marcelino, Ador Ronald Angelo K. Muñoz, Hendrik S. Onson, Bea Laurice P. Osorio, Holden Ian D. Peralta Group 7 2G Medical Technology Biological Chemistry Laboratory

ABSTRACT
RNA was isolated from yeast (Saccharomyces cerevisiae) by heating the active dry yeast with alkaline NaOH. This method of RNA extraction involved the disruption of the cell membrane and subcellular nucleus to break open and discharge the nucleic acids. RNA was extracted from associated proteins with HCl extraction and was treated with ethanol and ether to remove lipids. The absorbance of the isolated RNA was measured at 260 nm and 280 nm and underwent hydrolysis for characterization. The hydrolyzed RNA was characterized by different tests: test for ribose, test for phosphate, test for purines and test for pyrimidines. A dark green solution, yellow crystalline precipitate, reddish brown residue and a violet precipitate were the positive results obtained from each tests respectively.

INTRODUCTION
Ribonucleic acid (RNA) is one of the three major macromolecules that are essential for all known forms of life. Like DNA, RNA is made up of a long chain of components called nucleotides. Each nucleotide consists of a nucleobase (sometimes called a nitrogenous base), a ribose sugar, and a phosphategroup. The sequence of nucleotides allows RNA to encode genetic information. For example, someviruses use RNA instead of DNA as their genetic material, and all organisms use messenger RNA(mRNA) to carry the genetic information that directs the synthesis of proteins. Like proteins, some RNA molecules play an active role in cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these active processes is protein synthesis, a universal function whereby mRNA molecules direct the assembly of proteins on ribosomes. This process uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal RNA (rRNA) links amino acids together to form proteins. The chemical structure of RNA is very similar to that of DNA, with two differences--(a) RNA contains the sugar ribose while DNA contains the slightly different sugar deoxyribose (a type of ribose that lacks one oxygen atom), and (b) RNA has the nucleobase uracil while DNA contains thymine (uracil and thymine have similar base-pairing properties). Unlike DNA, most RNA molecules are singlestranded. Single-stranded RNA molecules adopt very complex three-dimensional structures, since they are not restricted to the repetitive doublehelical form of double-stranded DNA. RNA is made within living cells by RNA polymerases, enzymes that act to copy a DNA or RNA template into a new RNA strand through processes known as transcription orRNA replication, respectively. Nucleotides have three characteristic components: (1) a nitrogenous base, (2) a pentose sugar and (3) a phosphate. [2] Nitrogenous bases are derived from two parent compounds, purines and pyrimidines. Both DNA and RNA have purine bases, adenine and guanine, as well as a major pyrimidine base cytosine. However, they differ in the second major pyrimidine base that binds with adenine, thymine in DNA and uracil in RNA. Another major difference between DNA and RNA is their sugar components. 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. The objectives of this experiment are to isolate RNA from yeast where it can be assessed of its purity with UV measurement and to characterize and identify the principle involved in the reactions of RNA with different tests (test for ribose, test for phosphate, test for purines, and test for pyrimidines) following hydrolysis.

Figure 1. A nucleotide consists of a pentose sugar, a phosphate group and a nitrogenous base.

RESULTS AND DISCUSSION Alkaline Hydrolysis In the hydrolysis of RNA. The solution was cooled and was mixed with 1 ml ammonium molybdate solution and 10 ml water. Chemical Characterization Chemical Test Test for Ribose Test for Phosphate Test for Purines Std. The reaction product is an equimolar mixture of 2'. The mixture was heated over a small flame shaking frequently until the contents of the tube turned brown. This reaction depends on the conversion of ribose to an aromatic aldehyde (furfural) which then reacts with Orcin (3. 1N KOH.3N NaOH. Litmus: Red to Blue RNA from yeast Light orange solution Light yellow sol¶n Yellowish coloration White turbid sol¶n with white ppt. The group failed in getting a positive in the RNA of yeast because the conversion of ribose to an . concentrated HNO3. Few drops of concentrated HNO3 were added to the solution and evaporated to dryness on a hot plate. The mixture was heated in a water bath for 60 minutes. Test for Ribose To test for ribose. concentrated H2SO4. solution Light Green sol¶n Light yellow sol¶n Brownish Red coloration Formation of purple and white ppt. Few drops of water were added to the dried solution and were again evaporated leaving a reddish brown residue as a positive result. The mixture was cooled before adding 0. Ba(OH)2 and orcinol reagent. The formation of a violet precipitate was observed. ammonium molybdate. Test for Pyrimidines(Wheeler-Johnson Test) An excess of bromide water was added to 0.5 ml RNA solution until the solution turned yellow. An excess of Barium Hydroxide was added to the solution and tested with litmus paper. The colorless liquid was added to 1 ml water and was placed on a water bath for 5 minutes. The mixture was heated on water bath for 5-10 minutes.and 3'-nucleoside monophosphates. Procedure Alkaline Hydrolysis Two millilitres of 0. The group succeeded to get a positive result for the standard solution.5 ml concentrated HNO3 and was placed over a flame until white fumes appeared. The solution was boiled on a hot plate until a change in color to light yellow or colorless occurred. Test for Purines (Murexide Test) Five drops of RNA solution was placed in a small evaporating dish. the orcin reaction was used. Reagents The following reagents were used for alkaline hydrolysis and characterization of RNA were 0. B. The solution was let to stand for 5 minutes before noting a yellow crystalline precipitate as a positive result.EXPERIMENTAL A. Table 1. the 2'OH group in ribonucleotides renders RNA susceptible to strand cleavage in alkali solutions. Litmus: Red to Blue (+) result Blue green sol¶n Yellow crystalline ppt Brown red residue Test for Pyrimidines Purple ppt Characterization tests are used to describe the reactions of RNA to different reagents in order to exemplify its structural features.5dihydroxy toluene) to form an aldehyde-phenol condensation product that is blue-green/dark green in color.3N NaOH was added to a small amount of RNA isolate placed in a test tube covered with marble. 10% KOH.5 ml hydrolyzed RNA solution was mixed with 2 ml Orcinol reagent. 0. Test for Phosphate One millilitre concentrated H2SO4 was added to 1 ml RNA solution and to 1 ml standard phosphate solution. The residue formed was moistened with 10% KOH and heated to dryness. Test for Ribose In the test for the presence of ribose. The hydrolyzate was cooled and the pH was adjusted to pH 4-6 with glacial acetic acid using pH paper. A dark green coloration was obtained as a positive result. The net effect of this reaction is to transfer a phosphate from one nucleotide to the adjacent nucleotide in the chain. bromine water.

It was due faulty time management. or commonly known as murexide test. the sample is treated with bromine water to form 5-bromo-6hydroxyhydro derivatives which produces a yellow coloration. the RNA is reacted with nitric acid since purines are known to be readily soluble in dilute acids.html http://people..hofstra. pp. The group failed yielding the redish brown coloration for the RNA of yeast. 3rd ed.scribd. 164-166.org/doi/abs/10. Harper¶s Biochemistry. . (2000). D. This is due to the reaction of ammonium molybdate solution which when dropped upon a sample. M. The group did not get the yellow precipitate for the standard solution right away. pp. pp. M. New York: Litton Educational Publishing Inc. Concentrated nitric acid oxidized it leaving a yellow precipitate upon evaporation. indicates the presence of phosphate by a yellow stain or a crust of yellow phospho-ammonium molybdate. However.de/Fakultaeten/nat_Fak_IV/Organisc he_Chemie/Didaktik/Keusch/p32_rib_rna-e.T. REFERENCES From books: Murray.mun.acs. The reaction of ribose with orcinol reagent to give a blue green condensation product.L. (1988).1021/jo01083a00 3 01/ 1959 http://www. The addition of barium hydroxide Ba(OH)2 gives a 5. 383386. which is a positive result for the presence of uracil in RNA. it forms a 5-bromo derivative. Test for Phosphate In the test for the presence of phosphate in RNA. Nelson.R. and Dalton. and Cox. New York: Worth Publishers. 345-346. Internet sources: http://www.K.com/doc/25162486/RNAPurification-and-Analysis Figure 2. the group obtained the yellow like solution.M. a violet precipitate.ca/biochem/courses/3107/Lectu res/Topics/bases_and_chains. One of the reasons is that the group did not added enough concentrated HNO3 For the RNA from the yeast. Lehninger Principles of Biochemistry.uniregensburg. It is also due to the carelessness of the group.aromatic aldehyde was not fully develop. 325-328.edu/beverly_clendening/Ad v_Molecular_Biology/Protocols/UV_Spec_Analysis _RNA&DNA. The group waited until the next meeting and obtained the positive result. it turned red when moistened with a base.htm 05 / 07 / 2003 http://pubs. 21st ed. which is a positive result for presence of purine bases (guanine or adenine). Organic Chemistry in the Laboratory. Test for Purines (Murexide Test) In the test for purines. Connecticut: Appleton & Lange. D. R.htm http://www. Upon dehydration in solution. Yip. (1979). 5-dibromo6-hydroxyhydro derivatives. a yellow precipitate is obtained. Test for Pyrimidines(Wheeler-Johnson Test) In the test for pyrimidines.

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