LAB - 9 Nucleic acids

Aim of the class: studies on the composition and some properties of nucleic acids Nucleic acids are composed of nucleotides. Each nucleotide contains an organic base, sugar compound (ribose or deoxyribose) and phosphate. Two types of organic bases occur in nucleotides. Some of them contain purine (adenine or guanine), while the others contain pyrimidine (cytosine, uracil or thymine). Each base binds to ribose or deoxyribose by N--glycoside bond, whereas the phosphate is bound to sugar compound through ester bond with OH group at carbons 3or 5 of ribose or deoxyribose. Individual nucleotides are connected with phosphodiester bonds between carbons 3 and 5. Acidic character of nucleic acids originates from phosphate residues. Each of them contains proton (H+), which is able to dissociate in physiological pH. For this reason nucleic acids are polyanions. They are carriers of numerous negative electric charges. It makes them able to interact with polycations, especially with basic proteins, which are carriers of positive electric charges. Furthermore, nucleic acids may bind some basic substances of low molecular weight, e.g. Methylene Blue. In natural conditions DNA binds mainly to basic proteins, called histones, whereas RNA binds mainly to neutral proteins, which are constituents of ribosomes. Complexes of nucleic acids with proteins are called nucleoproteins. In student laboratory, some artificial nucleoproteins may be obtained by mixing the nucleic acids with blood serum. Nucleoproteins dissociate in concentrated salt solutions releasing nucleic acids and proteins. In alkaline medium the nucleic acids form salts, which are referred to as nucleates. They are easily soluble in water and may be precipitated from the solution with ethanol. 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 (or deoxyribose) and phosphate. In the same conditions the pyrimidine nucleotides are stable and do not undergo further hydrolysis. Purine bases may be easily precipitated from acidic hydrolysates as insoluble complexes with copper (Cu+) or silver (Ag+) ions. Ribose submitted to the action of concentrated HCl at 100oC is dehydrated forming furfural, which reacts with orcin (in the presence of Fe3+ ions) forming a complex with stable green colour. Deoxyribose (contained in DNA) is converted into hydroxylewulinic aldehyde when heated in concentrated H2SO4. This aldehyde reacts with diphenylamine forming blue colour complex. Final product of purine metabolism in human body is uric acid, which is a hardly soluble substance. Sodium salts of uric acid (sodium urate) demonstrate a higher solubility, whereas ammonium, copper and silver urate demonstrate very low solubility in water. Uric acid demonstrates weak reducing properties. Uric acid is converted into alloxan and urea under the action of nitric acid (V). The condensation of two alloxan molecules with NH3 results in formation of murexic acid, which reacts with NH4+ or Na+ forming colour (purple or blue, respectively) product: murexide. Reducing properties of uric acid make this substance an important biological antioxidant, which inactivates reactive oxygen species (ROS) and prevents formation of ROS.
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Laboratory procedures
1. Isolation of nucleic acids from yeast Suspend 25 g of baker yeast in 30 ml of 10% NaCl, homogenise them with the use of a glass rod, add 30 drops of Phenol red dissolved in ethanol and neutralise the suspension first with 10% NaOH and then with 2% NaOH until red-orange colour appears. Heat the suspension in a boiling water bath for 30 minutes with occasional mixing. In these conditions the nucleic acids are converted into sodium nucleate, which become soluble in the extracting solution. Cool the mixture and separate the insoluble compounds by centrifugation or filtration through a paper filter. Collect the supernatant (or filtrate), which contains nucleic acids (in a form of sodium nucleate) and discard the sediment. Add 3 volumes of ethanol and leave the mixture for 15 minutes. The nucleic acids precipitate from the solution. Separate the precipitate by centrifugation or filtration and redissolve it in 15 ml of 0.1M NaOH. Such a specimen of nucleic acids (sodium nucleate) will be used for further studies. 2. Solubility of nucleic acids a. Prepare 1 ml sample of sodium nucleate solution and supplement it with a few drops (drop by drop) of 2M HCl. Adding of HCl results in opalescence of the solution and precipitation of nucleic acids. Add similar volume of 2 M NaOH (drop by drop). The precipitate dissolves and opalescence disappears. b. Prepare 1ml sample of sodium nucleate solution and supplement it with 2 ml of ethanol. Observe precipitation of sodium nucleate. 3. Precipitation of nucleic acids with protein solutions Prepare two test tubes (A and B). Add 1 ml of sodium nucleate to tube A and 1 ml of water to tube B. Supplement both tubes contents with 2-3 drops of diluted (1: 20) acetic acid and 1 ml of diluted (1:10) blood serum. Observe a precipitation of artificial nucleoprotein in tube A containing sodium nucleate. Such a precipitate does not appear in tube B (containing water). Then add 2 ml of saturated NaCl solution into both tubes. Observe dissolution of precipitated nucleoprotein in tube A and precipitation of serum proteins in tube B. 4. Precipitation of nucleic acids with Methylene Blue Prepare 1 ml sample of sodium nucleate solution and supplement it with 2 3 drops of diluted (1:20) acetic acid and a few drops of 1% Methylene Blue. Observe colour salt precipitation of the basic dye with nucleic acids. 5. Nucleic acid sugar compounds detection The sugar compounds (ribose and deoxyribose) may be detected directly, without previous hydrolysis of nucleic acids. The tests detecting sugar compounds may be performed on 0.1% solution of sodium nucleate (nonhydrolysed).

a. Molisch test a general test detecting sugars Prepare 1 ml sample of 0.1% sodium nucleate, add 1 drop of 10% -naphtol (dissolved in ethanol) and supplement the mixture with 0.5 ml of concentrated H2SO4 on the wall of the tube. The heavy H2SO4 solution flows down to the bottom of the test tube and the mixture of nucleate with -naphtol rises up, forming two-phase arrangement of both solutions. Observe red-violet layer at the contact place of both phases. The colour spreads after a gentle mixing the tube content, taking in the whole mixture. b. Detection of ribose - orcin test Prepare 1 ml sample of 0.1% sodium nucleate and add 1 ml of orcin reagent (orcin with FeCl3 dissolved in concentrated HCl). Mix the tube content by a gentle shaking and put the tube into a boiling water bath. Observe an appearance of green colour product of ribose orcin interaction. c. Detection of deoxyribose diphenylamine test Prepare 1 ml sample of 0.1% sodium nucleate and add 2 ml diphenylamine reagent (diphenylamine dissolved in a mixture of concentrated acetic and sulphuric acids). Mix the tube content by a gentle shaking and put it into a boiling water bath for 10 minutes. Observe an appearance of blue colour product of deoxyribose diphenylamine interaction. 6. Detection of purine bases in nucleic acids Nucleic acids hydrolysis Prepare 4 ml sample of 0.1% sodium nucleate and add 1 ml of 2.5M H2SO4 solution. Mix the test tube content and put it into a boiling water bath for 1 hour. Hydrolysis of N--glycoside bonds between purine bases and ribose or deoxyribose results in a release of purine bases (adenine and guanine). Cool the hydrolysate to room temperature and neutralise it with 2M NaOH (drop by drop, under the control of litmus-paper) to achieve a weak acidic pH. Use the hydrolysate for detection of purine bases. a. Detection of purines by precipitation with Cu+ ions Prepare 1 ml sample of hydrolysate and heat it to boiling. Add a few drops of CuSO4 solution (containing Cu2+) and supplement the mixture (drop by drop) with saturated NaHSO3 solution. The NaHSO3 reduces Cu2+ to Cu+. Interaction of purines with Cu+ results in precipitation of insoluble yellowwhite complex. Repeat the same test with water instead of nucleic acid hydrolysate. No precipitation is observed. b. Detection of purines by precipitation with Ag+ ions Prepare 1 ml sample of hydrolysate, add a few drops of AgNO3 and 1 ml NH4OH solution. Observe precipitation of complexes of purines with Ag+ ions. Repeat the same test with water instead of nucleic acid hydrolysate. No precipitation is observed. 7. Detection of phosphate Add to test tube in order: 3 drops of 0.1% sodium nucleate, 2 ml of distilled water, 1.5 ml of 10% TCA, 0.5 ml of molybdic reagent and 0.5 ml of eiconogene reagent and mix. Observe blue colour appearance, characteristic for phosphate presence in solution.
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8. Some properties of uric acid a. Detection of uric acid murexide test Prepare 1ml sample of disodium urate (disodium salt of uric acid) in a small evaporating dish and add a few drops of nitric acid. Evaporate the mixture to obtain dry red colour sediment of murexic acid. Its salts are named murexides. Apply a drop of NH4OH solution on a margin of the sediment and observe purple colour of ammonium murexide. Apply a drop of NaOH solution on the opposite margin of the sediment and observe blue colour of sodium murexide. b. Solubility of uric acid salts Prepare four samples of 1ml disodium urate and supplement them (drop by drop) with 2M solutions of HCl, NH4Cl, CuSO4 and AgNO3, respectively. The soluble disodium urate changes into hardly soluble or insoluble products, which precipitate from the solution forming insoluble sediments. They are: uric acid, ammonium urate, copper urate and silver urate, respectively. c. Reducing properties of uric acid Prepare 1 ml sample of disodium urate and add 0.5 ml of Folin reagent (containing sodium tungstate in phosphoric acid). Alkalise the mixture with 0.5 ml of 2M NaOH. Uric acid reduces sodium tungstate contained in Folin reagent forming blue colour wolfram oxides (WO2 x 3WO3).

Laboratory task
Check if the tested solution contains ribose, deoxyribose or uric acid?