Biochem-M10-Notes - biochem nucleic acid PDF

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MLS 5a | BIOCHEMISTRY FOR MLS (Lab) Jovero, Jocel Therese C. MLS 2-AMODULE 10: NUCLEIC ACIDSLEARNING OUTCOMES Test the properies of prepared Ribonucleic Acid (RNA) Appreciate the importance of nucleic acids in daily life aciviies Prepare nucleoproteins in the laboratory and determine their test resu...

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MLS 5a | BIOCHEMISTRY FOR MLS (Lab) MODULE 10: NUCLEIC

Jovero, Jocel Therese C. MLS 2-A

ACIDS

LEARNING OUTCOMES 1. Test the properties of prepared Ribonucleic Acid (RNA) 2. Appreciate the importance of nucleic acids in daily life activities 3. Prepare nucleoproteins in the laboratory and determine their test results 4. Evaluate the constituents of nucleoproteins 10.1 NUCLEIC ACIDS Nucleic acids are found in all living cells. They play a vital role in cellular organization and function. They are essential in cell division, reproduction, and transmission of hereditary factors. They are also involved in protein synthesis of different tissues. They are a component of molecules that function as co-factors and co-enzymes. Nucleic acids are mildly soluble in cold water, insoluble in alcohol, but mixes instantly in weak alkali metal salts. They are precipitated from alkaline solution by the addition of acid. Complete hydrolysis of nucleotides yield a mixture of sugar (ribose), purine (adenine and guanine) or pyrimidine bases (thymine, cytosine and uracil), and phosphate. In preparing RNA from yeast, heating with alkali (10% NaOH) is essential. This extracts the nucleic acid and water soluble proteins, and inactivates the nucleases. The nucleic acid is separated from associated protein and other interfering substances by acid extraction at pH 4-5. The final step is treatment with alcohol and concentrated HCl to precipitate the RNA, followed by 6N acetic acid by repeated washing with 95% alcohol and other organic solvents to remove substances that may interfere with the chemical tests. Preparation of RNA from yeast (USA Biochemistry Laboratory Manual) 1. Place 10 g of dried yeast in a beaker. 2. Add 15 mL of 10% NaOH with 85 mL of water. 3. Heat the mixture in a water bath for 30 min with frequent stirring. After heating, filter and allow the filtrate to cool. Vacuum filtration can also be used. 4. Add 6N acetic acid until faintly acid to litmus paper and then filter again. 5. Evaporate the solution to ½ its original volume by heating again in a water bath. Filter if necessary and allow to cool. 6. Pour with vigorous stirring into the beaker containing 30 mL of 95% ethyl alcohol with 8 drops of concentrated HCl. This is yeast RNA. 7. Allow the yeast RNA t o settle, and decant or remove the upper supernatant liquid with a medicine dropper. 8. Add 2 mL of 95% alcohol to the precipitate. 9. Shake and remove the alcohol using a medicine dropper. Waste disposal: Organic Non-halogenated Waste A nucleoprotein is a conjugated protein consisting of a protein linked to a nucleic acid, either DNA or RNA. The protein combined with DNA is commonly either histone or

protamine; the resulting nucleoproteins are found in chromosomes. Many viruses are little more than organized collections of deoxyribonucleoproteins or ribonucleoproteins. Little is known about the proteins linked with RNA; unlike protamine and histone, they appear to contain the amino acid tryptophan. Preparation of Nucleoproteins (USA Biochemistry Laboratory Manual) 1. Place 10 g compressed yeast in a mortar. 2. Sprinkle a small amount of sand and add 5 mL ether and 10 mL distilled water. 3. Triturate thoroughly. During trituration, add now and then 1 mL water until the mixture becomes more fluid (the whole process can be completed in 5 min). (The ether kills the yeast so the cell is more thoroughly comminuted or broken into smaller pieces). 4. Transfer the liquid into a 500-mL Erlenmeyer flask, and add 0.4% NaOH to a final volume of about 125 mL (The alkali extracts the nucleoproteins together with the water-soluble proteins of the yeast). 5. Add a little toluene and allow to stand with frequent shaking for 30 min. 6. Filter using cheesecloth. 7. On the filtrate, add one drop at a time of 10% HCl while stirring thoroughly, and continuously add more as long as the milkiness increases; the protein completely separates and the liquid is practically clear. 8. Filter using cheesecloth and retain the precipitate on the filter for nucleoprotein tests. Reserve the filtrate for subsequent tests. Waste disposal: Organic Non-halogenated waste 10.2 MODULE 10 EXPERIMENT Part A. Chemical Tests for the Components of Nucleic Acids YouTube Video: Chemical Tests for the Components of Nucleic Acids https://youtu.be/8B5ZIxIIT4g

Nucleic acids are molecules that are involved in heredity. Nucleic acids are the macromolecules involved in one of the most important process in a living organism. The ability to transfer anatomical and biochemical characteristic from one organism to another which is known as heredity. There are two types of nucleic acids, DNA and RNA. In today’s experiment, we will be using RNA derived from yeast. Test for Solubility Solvents used:  Dil. NaOH  Ethanol  Dil. HCl  Hot H2O

 Cold H2O

Procedure: 1. Place a pinch of yeast RNA in a five separate test tubes 2. Add 2 mL of each solvent to the corresponding test tubes Result: Insoluble cold water, ethanol, dil. HCl

MLS 5a | BIOCHEMISTRY FOR MLS (Lab) MODULE 10: NUCLEIC

ACIDS

Slightly soluble  hot water Soluble  dil. NaOH *Test for the Chemical Components of Nucleic Acids Nucleic acids are polymers of nucleotides. The nucleotide monomer is composed of nitrogenous base, pentose sugar, and a phosphate group. There are numerous test that can be used to determine the presence of these components. But before conducting these experiments, it is very necessary that the RNA should undergo hydrolysis first. Hydrolysis would break down the nucleic acid into three nucleotides and the nucleotides to its free components. Hydrolysis is conducted by placing a pinch of yeast RNA in a test tube. Then, mixed it with 10 mL of 10% H 2SO4 solution. Then, heat the mixture to boiling for 2 minutes. Cool the mixture after boiling. This is known as the hydrolysate. Test for Purine 1. Put 1mL of the hydrolysate in a separate test tube 2. Add 5 drops of ammoniacal silver nitrate 3. Add 1mL of conc. ammonium hydroxide solution 4. The presence of a white precipitate is a positive result for the test for purine Test for Pentose Sugar 1. Place 1mL of hydrolysate into a separate test tube 2. Mixed it with two drops of Molisch Reagent 3. Tilt the test tube at 45 degree angle and superimpose 1mL of conc. sulfuric acid along the side. 4. The formation of the purple ring indicates the presence of a carbohydrate. In the case of nucleic acid, the carbohydrate is in the form of a pentose sugar. Test for Phosphate Group 1. Place 1mL of hydrolysate in a separate test tube 2. Mix with 3mL of ammonium hydroxide solution 3. Add 10% of nitric acid solution 4. Continue the addition of nitric acid until the solution will give an acidic reaction to litmus paper. 5. After the color change in litmus paper, add 3mL of ammonium molybdate solution 6. Heat the mixture to boiling for 2 minutes 7. The formation of yellow precipitate indicates the presence of a phosphate group

Jovero, Jocel Therese C. MLS 2-A...