NUCLEIC ACIDS

Nucleic acids are polyanionic molecules of high molecular weight. These polymers are composed of a sequence of sub-units of nucleotides so that the whole is usually termed as polynucleotide (Zaballa et al., 2010). According to Calano et al. (2011), there are two types of nucleic acids, the ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) which hydrolysis yield the sugar ribose and deoxyribose, respectively. Nucleic acids are found in the chromatin granules of the nucleus, nucleolus and cytoplasm. They are responsible for the storage and transmission of genetic information. Nucleic acids are mostly conjugated with proteins such as histones to form nucleoproteins, which on hydrolysis yield protein and nucleic acids. DNA can be isolated from thymus or muscle tissues, RNA from yeast (Nucum, 2007). Nucleic acids are composed of nucleotides. Each nucleotide contains an organic base, sugar compound (ribose or deoxyribose) and phosphate (Apps et al., 1992). One important nucleotide is adenosine monophosphate (AMP). It is formed by the reaction of adenosine (a nucleotide) with one molecule of phosphoric acid. If two phosphate groups react with adenosine, adenosine diphosphate (ADP) is formed; ATP, adenosine triphosphate, is formed when three phosphate groups react. ATP is involved in various metabolic processes including the storage and release of energy from its phosphate bond (answer to Question 4). DNA is made up of long chain of nucleotides. If the proper sequence of its nucleotides is changed, mutation will occur. Mutation may occur due to exposure to radiation or certain chemicals. This may result to cancerous cells. In a study by

Macgregor and Squire (2002), they said that microarray analysis may provide invaluable information on disease pathology, progression, resistance to treatment, and response to cellular microenvironments and ultimately may lead to improved early diagnosis and innovative therapeutic approaches for cancer. The main difference between DNA and RNA lies in their sugar components and in one of their pyrimidine bases. DNA contains deoxyribose and thymine while RNA has ribose and uracil instead of thymine (Rafelson et al.1980). Various physicochemical studies on DNA have shown that on aqueous solution, DNA has a very large molecular weight and very rigid structure (Zaballa et al., 2010). Hence, DNA exhibits high viscosity. However, with increasing temperature, the viscosity of DNA decreases due to the disruption of its very rigid structure (answer to Question 5).

Objectives: 1. To isolate RNA from yeast, 2. To prepare acid-hydrolyzed samples of RNA, and 3. To determine composition of nucleic acids (RNA) through qualitative tests

Results and Discussion:

Isolation of RNA from yeast 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 with the grinding with sand.

Test for Nucleoproteins According to Sackheim and Lehman (1998), nucleoproteins are proteins combined with nucleic acids. Since they have protein in their structure therefore, they undergo all the reactions that proteins do. One of the protein reactions is the Biuret test. This test positively identifies the presence of proteins in solution with a deep violet color. Table 1. Results of Biuret Test Extract Observation Yeast purple w/ purplish white membranous precipitate

Do observations indicate presence of peptide bonds? Yes

Table 1 shows that the yeast extract give positive result to Biuret test. It strongly indicates that the extract contain a peptide bond, thus have a protein. Therefore, this clearly presents that the extract is a protein.

Mild Acid Hydrolysis

An action of 0.5M H2SO4 at 100oC results in hydrolysis of nucleic acids to mononucleotides. Purine nucleotides undergo further hydrolysis resulting in complete breakdown of N--glycoside bonds and ester bonds releasing free purine bases, ribose and phosphate (www.biochemia.amb.edu.pl/pdf/class9.pdf retrieved on March 3, 2012). Hydrolysis studies do not end on the hydrolysis reaction alone. It is also necessary to do qualitative tests on the products of hydrolysis to be able to determine what species are present. In the experiment, three common qualitative analyses were employed: test for phosphates, the Bials test, and the test for purine bases.

Test for the Presence of Phosphates Ribonucleic acid (RNA) is one of the three major macromolecules that are essential for all known forms of life. It 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 phosphate group (Nucum, 2007).

Table 2. Results of Test for the Presence of Phosphates Specie Hydrolysate Observation clear light yellow solution w/ yellow precipitate

For the test for the presence of phosphate in the hydrolyzed RNA, as shown in Table 2, the positive result is a yellow precipitate. The positive result is because of the reaction of ammonium molybdate solution, which when dropped onto the sample will result to a yellow stain which indicates the presence of phosphate or a crust of phospo-ammoniummolybdate.

Test for the Presence of Ribose Bials test is a test for the presence of pentose. It can be used as a test for RNA due to the presence of ribose. Bials reagent consists of reagents which promotes the dehydration of ribose to furfural. 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 is used as the dehydrating acid and orcinol as the condensation reagent. A positive test for pentose is indicated by a blue or green condensation product (answer to Question 3). Table 3. Results for Bial Orcinols Test Test solution Observation
Do observations indicate presence of pentose sugar?

Hydrolysate

dark blue green solution w/ dark blue precipitate

yes

For the Bials test, the hydrolysate yields a positive result. The result indicates the presence of a pentose sugar and it can be said that this is ribose (Table 3).

Test for the Presence of Purines

The heterocyclic bases present in nucleic acids are divided into two types purines and pyrimidines (Calano et al., 2011). The two purines found in nucleic acids (both DNA and RNA) are adenine and guanine (answer to Question 1). Nucleotide bases such as purines are relatively insoluble in water. They may exist in two or more tautomeric forms. Purine bases may be easily precipitated from acidic hydrolysates as insoluble complexes with silver (Ag+) ions in ammoniacal silver nitrate (answer to Question 2). A positive result is indicated by the presence of a white precipitate.

Table 4. Results of Test for Presence of Purines Test solution Observation

Do observations indicate presence of purines?

Hydrolysate

Formation of whitish membranous solid in brown solution

yes

For the test for purine bases as presented in Table 4, the hydrolysate gives a positive result. This indicates that purines are present in the hydrolysate.

Conclusion: Nucleic acids are mostly conjugated with proteins to form nucleoproteins. Hydrolysis of the nucleoproteins yields proteins and nucleic acids. Further hydrolysis gives phosphates, pentose sugars (ribose or deoxyribose) and nitrogen bases (pyrimidines and purines).