CHEMICAL CHARACTERIZATION AND ALKALINE HYDROLYSIS OF RIBONUCLEIC ACID FROM YEAST

Erika I. Pineda, Marc Delvin C. Quero, Ryan R. Querubin,Rafaelle Angelica A. Raspado and Aimee Liza S. Ravelo

Group 8 2G Medical Technology Biochemistry Laboratory

ABSTRACT

This experiment was conducted to determine the behaviour of Ribonucleic Acid (RNA) towards various qualitative colour reaction tests following alkaline hydrolysis. The sample RNA was isolated from yeast (Saccharomyces cerevisiae). A portion of the isolated RNA was subjected to alkaline hydrolysis using 0.3 M NaOH. The RNA hydrolysate was characterized by different tests: Test for Ribose, Test for Phosphate, Murexide Test for Purines and Wheeler-Johnson Test for Pyrimidines. A peach solution, yellow crystalline precipitate, orange residue and a basic turbid solution were obtained from each test respectively. The results from the reaction of the RNA hydrolysate towards the different tests were compared to the positive results obtained from that of a standard RNA sample. Standards served as a basis whether the extracted RNA sample gave the expected results. The hydrolysate was positive in Test for Phosphate, Murexide Test and Wheeler-Johnson Test. A negative result was obtained from the Bial-Orcinol’s Test for Ribose.

I. 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 cell’s 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. [1] 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.[9] 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. [1]

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]

This experiment aims to qualitatively characterize the structure and composition of RNA from baker’s yeast following hydrolysis. Since the isolated RNA sample was subjected to hydrolysis, it is but proper to compare the behaviour of the hydrolysate to that of the standard solutions. Standards served as a basis whether the extracted RNA sample gave the expected results. Also, this part of the experiment identifies the principle involved in the reactions of RNA with test for ribose, test for phosphate, Murexide Test for Purines and Wheeler-Johnson Test for Pyrimidine bases.

II. EXPERIMENTAL

A. Compound Tested

Baker’s Yeast (Saccharomyces cerevisiae)

B. Procedure

Alkaline Hydrolysis

Two millilitres of 0.3 M NaOH was added to a small amount of RNA isolate placed in a test tube. The mixture was placed in a boiling water bath for 60 minutes. Prior to heating, the test tube containing the mixture was covered with a marble. The hydrolysate was cooled and the pH

was adjusted to pH 4-6 with glacial acetic acid using pH paper.

Test for Ribose

Two millilitres of orcinol reagent was added to 0.5 mL hydrolyzed RNA solution. Same amount of orcinol reagent was added to a 0.5 mL standard ribose solution in a separate test tube. The mixtures were heated on a water bath for 5-10 minutes. Any change in colour was noted.

Test for Phosphate

One millilitre concentrated H2SO4 was added to 1 mL RNA solution and to 1 mL standard phosphate solution. Each mixture was placed in an evaporating dish. Afterwards, each mixture was heated over a small flame shaking frequently until the contents turned brown. Each mixture was cooled before adding 0.5 mL concentrated HNO3 and was placed over a flame until white fumes appeared. The colourless 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 left to stand for 5 minutes. Any change in colour was noted.

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. Same procedure was done to the standard guanine and adenine solution. Any change in colour was noted.

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 colour to light yellow or colourless occurred. An excess of Ba(OH)2 was added to the solution and tested with litmus paper. Any change in colour was noted.

III. RESULTS AND DISCUSSION

In the base catalyzed hydrolysis of a nucleic acid, the hydroxyl ion assists the attack of the 2’ hydroxyl group on the phosphorus leading to the formation of the cyclic 2’,3’ monophosphate intermediate. The intermediate is highly unstable.

Introduction of water to form either 2’ or 3’ nucleosides as products stabilizes the said species. Only RNA undergoes base hydrolysis. The phosphodiester bond between two ribonucleotides can be broken by alkaline hydrolysis because of the free 2' hydroxyl group. [7] Figure 1 shows the location of the 2’-hydroxyl group of RNA which is a major difference between RNA and DNA. At high pH, RNA is degraded because there is deprotonation of the nitrogenous bases.

Figure 1. The 2’-hydroxyl group of RNA

Figure 2. Base-catalyzed Hydrolysis of RNA

Characterization Tests are employed to describe the reactions of RNA to various reagents in order to exemplify its structural features.

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

Bial-Orcinol’s test is a test for the presence of pentose. It can be used as a test for RNA due to the presence of ribose. Bial’s reagent consists of reagents which promotes the dehydration of ribose to furfural (Figure 3). The presence of furfural can be detected by the addition of a test reagent, known as the condensation reagent. The condensation reagent is generally a phenolic compound that reacts with furfural to give a highly-colored product. In the experiment, hydrochloric acid was used as the dehydrating acid and orcinol (5-Methylbenzene-1,3-diol) as the condensation reagent. A positive test for pentose is indicated by a blue or green condensation product. The standard ribose solution demonstrated a positive result in the experiment. However, the RNA hydrolysate was negative mainly due to poor or incomplete purification method that was employed.

Figure 3. Formation of furfural form ribofuranose.

Figure 4. The reaction of Ribose with Orcinol Reagent.

In the test for the presence of phosphate in both standard phosphate solution and RNA hydrolysate, a yellow precipitate was 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. Formation of yellow crystals followed. Both were positive in the Test for Phosphate. This indicates that a phosphodiester bond (Figure 4.) between the 3’ Carbon atom and 5’ Carbon atom of the ribose sugar.

Figure 5. The Phosphodiester bonds between the 3’ Carbon and 5’ carbon Atom.

In the test for purines (Figure 6), 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. The yellow coloration became red when moistened with a base which is a positive test for the presence of adenine and guanine.q However, only those which underwent acid hydrolysis should give a positive result. Base hydrolysis acts on the cleavage of the phosphodiester bond and not on the purine N-glycosyl bonds. Therefore, both the standard solution which is unhydrolyzed and the base-hydrolyzed RNA from yeast were negative.

Chemical TestStandard

RNA Solutions

RNA Hydrolysat

e

Test for RiboseBlue Green

SolutionPeach

SolutionTest for

PhosphateYellow

SolutionYellow

SolutionTest for Purines(Murexide Test)

Colorless Residue

Orange Residue

Test for Pyrimidines

(Wheeler-Johnson Test)

Purple Precipitate. Red Litmus

Paper turned Blue.

Turbid Solution.

Red Litmus Paper turned

Blue.

Figure 5. Nucleotide Bases

Figure 6. The reaction of RNA in Murexide Test.

In the test for Pyrimidines, the sample was treated with Bromine water to form 5-Bromo-6-Hydroxyhydro derivatives which produces a yellow coloration. Upon dehydration in solution, a 5-Bromo derivative was obtained. The addition of Ba(OH)2 gives a 5, 5-Dibromo-6-Hydroxyhydro derivatives, a violet precipitate, which is a positive result for the presence of Uracil (Figure 7) in RNA. The standard solution was positive while the hydrolysate failed to demonstrate the expected result.

Figure 7. The structure of Uracil

Figure 8. The reaction of Bromine with Uracil

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] Boyer, R. (2005). Concepts in Biochemistry, 3rd

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[2] Nelson, D.L. and Cox, M.M. (2000). Lehninger Principles of Biochemistry, 3rd Edition. New York: Worth Publishers, pp. 325-328, 345-346.

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

[4] RNA. Retrieved from http://virtualology.com/virtualsciencecenter.com/halloforganicchemistry/RNA-Ribonucleicacid.com/ 6:22 PM February 8, 2011

[5] Characterization of Nucleic Acids. Retrieved from http://www.docshare.com/doc/83332/Characterization-of-Nucleic-Acids 7:17 PM February 8, 2011

[6] Alkaline Hydrolysis of RNA. http://www.mun.ca/biochem/courses/3107/Lectures/Topics/bases_and_chains.html 7:24 PM February 8, 2011

[7] 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

[8] Bell, C., Douglass, T., et. al., (2001). Organic Chemistry Laboratory with Qualitative Analysis Standard & Microscale Experiments 3rd Edition. United States of America: Thomson Brooks/Cole. p348

[9] Murray, R.K. (1988). Harper’s Biochemistry, 21st ed. Connecticut: Appleton & Lange, pp. 383-386.