CHEMICAL CHARACTERIZATION OF RIBONUCLEIC ACID

Ma. Angelica C. Marcelino, Ador Ronald Angelo K. Muoz, 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.

EXPERIMENTAL
A. Reagents The following reagents were used for alkaline hydrolysis and characterization of RNA were 0.3N NaOH, 1N KOH, concentrated H2SO4, concentrated HNO3, ammonium molybdate, bromine water, 10% KOH, Ba(OH)2 and orcinol reagent. B. Procedure Alkaline Hydrolysis Two millilitres of 0.3N NaOH was added to a small amount of RNA isolate placed in a test tube covered with marble. The mixture was heated in a water bath for 60 minutes. The hydrolyzate was cooled and the pH was adjusted to pH 4-6 with glacial acetic acid using pH paper. Test for Ribose To test for ribose, 0.5 ml hydrolyzed RNA solution was mixed with 2 ml Orcinol reagent. The mixture was heated on water bath for 5-10 minutes. A dark green coloration was obtained as a positive result. Test for Phosphate One millilitre concentrated H2SO4 was added to 1 ml RNA solution and to 1 ml standard phosphate solution. The mixture was heated over a small flame shaking frequently until the contents of the tube turned brown. The mixture was cooled before adding 0.5 ml concentrated HNO3 and was placed over a flame until white fumes appeared. The colorless liquid was added to 1 ml water and was placed on a water bath for 5 minutes. The solution was cooled and was mixed with 1 ml ammonium molybdate solution and 10 ml water. The solution was let to stand for 5 minutes before noting a yellow crystalline precipitate as a positive result. Test for Purines (Murexide Test) Five drops of RNA solution 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 and were again evaporated leaving a reddish brown residue as a positive result. Test for Pyrimidines(Wheeler-Johnson 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 Barium Hydroxide was added to the solution and tested with litmus paper. The formation of a violet precipitate was observed.

RESULTS AND DISCUSSION

Alkaline Hydrolysis In the hydrolysis of RNA, the 2'OH group in ribonucleotides renders RNA susceptible to strand cleavage in alkali solutions. The reaction product is an equimolar mixture of 2'- and 3'-nucleoside monophosphates. The net effect of this reaction is to transfer a phosphate from one nucleotide to the adjacent nucleotide in the chain. Table 1. Chemical Characterization
Chemical Test Test for Ribose Test for Phosphate Test for Purines Std. solution Light Green soln Light yellow soln Brownish Red coloration Formation of purple and white ppt. Litmus: Red to Blue RNA from yeast Light orange solution Light yellow soln Yellowish coloration White turbid soln with white ppt. Litmus: Red to Blue (+) result Blue green soln 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. Test for Ribose In the test for the presence of ribose, the orcin reaction was used. This reaction depends on the conversion of ribose to an aromatic aldehyde (furfural) which then reacts with Orcin (3,5dihydroxy toluene) to form an aldehyde-phenol condensation product that is blue-green/dark green in color. The group succeeded to get a positive result for the standard solution. The group failed in getting a positive in the RNA of yeast because the conversion of ribose to an

aromatic aldehyde was not fully develop. It is also due to the carelessness of the group.

REFERENCES From books: Murray, R.K. (1988). Harpers Biochemistry, 21st ed. Connecticut: Appleton & Lange, pp. 383386. Nelson, D.L. and Cox, M.M. (2000). Lehninger Principles of Biochemistry, 3rd ed. New York: Worth Publishers, pp. 325-328, 345-346. Yip, M.T. and Dalton, D.R. (1979). Organic Chemistry in the Laboratory. New York: Litton Educational Publishing Inc., pp. 164-166. Internet sources: http://www.uniregensburg.de/Fakultaeten/nat_Fak_IV/Organisc he_Chemie/Didaktik/Keusch/p32_rib_rna-e.htm 05 / 07 / 2003 http://pubs.acs.org/doi/abs/10.1021/jo01083a00 3 01/ 1959 http://www.mun.ca/biochem/courses/3107/Lectu res/Topics/bases_and_chains.html http://people.hofstra.edu/beverly_clendening/Ad v_Molecular_Biology/Protocols/UV_Spec_Analysis _RNA&DNA.htm http://www.scribd.com/doc/25162486/RNAPurification-and-Analysis

Figure 2. The reaction of ribose with orcinol reagent to give a blue green condensation product. Test for Phosphate In the test for the presence of phosphate in RNA, a yellow precipitate is obtained. This 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. The group did not get the yellow precipitate for the standard solution right away. The group waited until the next meeting and obtained the positive result. One of the reasons is that the group did not added enough concentrated HNO3 For the RNA from the yeast, the group obtained the yellow like solution. Test for Purines (Murexide Test) In the test for purines, or commonly known as murexide test, the RNA is reacted with nitric acid since purines are known to be readily soluble in dilute acids. Concentrated nitric acid oxidized it leaving a yellow precipitate upon evaporation. However, it turned red when moistened with a base, which is a positive result for presence of purine bases (guanine or adenine). The group failed yielding the redish brown coloration for the RNA of yeast. It was due faulty time management. Test for Pyrimidines(Wheeler-Johnson Test) In the test for pyrimidines, the sample is treated with bromine water to form 5-bromo-6hydroxyhydro 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-dibromo6-hydroxyhydro derivatives, a violet precipitate, which is a positive result for the presence of uracil in RNA.