EXPERIMENT NO. 9 HYDROLYSIS OF NUCLEIC ACIDS Abstract This experiment intended to study the acid and base hydrolysis of DNA and RNA. Furthermore, it aimed to characterize and differentiate the hydrolysates using various qualitative tests. Hydrolysis studies were done by the addition of HCl and NaOH to DNA and RNA samples. The systems were heated for 1 hour in a boiling water bath Control samples, consisting of unhydrolyzed DNA and RNA, were also prepared. After the hydrolysis procedure, Bial’s Test and test for purines were performed to characterize the hydrol hydrolysa ysates tes.. Despit Despite e a few errone erroneous ous result results, s, in genera generall the experi experimen mentt was succes successfu sfull lly y in provid providing ing knowl knowledg edge e regard regardin ing g nuclei nucleic c acid acid hydrol hydrolysi ysis. s. The The quali qualitat tative ive tests tests employ employed ed were were also also successful in differentiating the hydrolysates.
Discussion of Data and Results Nucleic acids are the most fundamental constituent of a living cell. They generally serve as the storehouses and carriers of genetic information. There are two types of nucleic acid: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), structures of which are as shown in figure 1.
Figure 1. Chemical structure of RNA and DNA. (http://scienceblogs.com/transcript/200 http://scienceblogs.com/transcript/2006/11/autism_rna.php 6/11/autism_rna.php))
As shown from the figure, monomer units of each nucleotide contain a five carbon sugar: ribose in RNA, and 2’- deoxyribose in DNA. The sugar units differ only in the 2’ hydroxyl group on the ribose. In the nucleic acids, two monomer units is connected through a phosphate residue attached to the hydroxyl of the 5’ carbon of one unit and the 3’ hydroxyl of the next one. This then forms a phosphodiester bridge between two sugar residues.
The backbone of nucleic acid is made up primarily of the phosphodiester-linked sugar residues. Each monomer carries a basic group, labeled as B in Figure 1, attached to the 1’ carbon of the sugar. The nucleic acid bases are of two kinds: the purines, adenine and guanine, and the pyrimidines, cytosine, thymine (for DNA), and uracil (for RNA). The structures structures of these bases are given in Figure 2.
Figure 2 . Nucleotide bases. (http://www.wvup.edu/ecrisp/fournucleotides.jpg)
DNA and RNA are polynucleotides. In principle, a polynucleotide could be generated from its monomers by elimination of water between each pair of monomers. This reaction is a condensation reaction. The reaction can be imagined as adding another nucleotide to the polynucleotide chain by a dehydration reaction. However, the reported free energy change is about +25 kJ/mol, a quite positive value. The equilibrium will then lie to the side where hydrolysis of the phosphodiester bond is favored. Though polynucleotides are thermodynamically unstable, their hydrolysis still proceeds at a very slow rate, unless catalyzed. Hydrolysis can either proceed by acid/base catalysis or through the action of enzymes. The site at which hydrolysis occurs could also differ. The sites of cleavage could be any of the following: cleavage in the sugar, in the phosphate backbone, or in the base. The reaction then may produce the following: purine and pyrimidine bases, oligonucleotides, nucleosides, ribose or deoxyribose and phosphates. In this experiment, the acid and base-catalyzed hydrolysis of DNA and RNA were studie studied. d. The The product products s of the hydrol hydrolysi ysis s reacti reaction on (or hydrol hydrolysa ysates tes)) were were also also charac character terize ized d and differentiated using Bial’s test and test for purine bases. From Figure 1, it can be noticed that each phosphodiester bridge carry a net negative charge. This net negative charge makes the nucleic acid less susceptible to nucleophilic species. Nonetheless, at alkaline conditions, base-catalyzed hydrolysis may occur and proceed as follows:
Figure 3. Base-catalyzed hydrolysis of RNA. [3]
In the base catalyzed hydrolysis of a nucleic acid, the hydroxyl ion assists the attack of the 2’ hydr hydrox oxyl yl grou group p on the the phos phosph phor orus us leadi leading ng to the the form format atio ion n of the the cycl cyclic ic 2’,3 2’,3’’ mono monoph phos osph phat ate e 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 absence of the 2’ hydroxyl group in DNA makes it unable to form the cyclic monophosphate intermediate. DNA is then immune to base hydrolysis. This property yet again contributes to the stability of DNA as a molecule. Chain cleavage of RNA can also be acid catalyzed. The products would still be a racemic mixture of 2’ and 3’ nucleosides. The reaction would proceed as follows:
Figure 4. Acid-catalyzed chain cleavage of RNA. [4]
From the figure, it can been that an acid-catalyzed chain cleavage also arrives at the cyclic 2’,3’monophosphate intermediate. This would then eventually rearrange to give the 2’ and 3’ nucleosides as products.
For DNA (also RNA) acid hydrolysis cleaves the predisposed purine N-glycosyl bonds. If the nucleic acids are placed in a dilute acid solution, coupled with heating, the adenine and guanine residues are liberated. What remains are apurinic sites. Mild heating doesn’t release pyrimidines. Further heating in sealed test tube or autoclave autoclave is required to cleave pyrimide pyrimide N-glycosyl N-glycosyl bonds. The acid-catalyz acid-catalyzed ed depurination of RNA/DNA proceeds as follows:
Figure 5 . Acid-catalyzed depurination of nucleic acids. [4]
In the reaction mechanism given in Figure 5, it can be deduced that the depurination occurs is promoted by the protonation of the purine base, thus, weakening the N-glycosyl bond. The Nglycosyl bond is then irreversibly broken by the neighboring oxygen atom giving a free pu rine base and an apuri apurinic nic nuclei nucleic c acid. acid. Acid-c Acid-cata atalyz lyzed ed depyri depyrimid midiza izatio tion n also also procee proceed d in a simil similar ar mechan mechanism ism as depuri depurinat nation ion.. Depyri Depyrimid midiza izatio tion n releas releases es cytosi cytosine ne and uracil uracil,, depend depending ing on the nuclei nucleic c acid acid being being considered. However, in acidic conditions, cytosine can be easily deaminated to uracil according to the following reaction:
Figure 6 . Acid-catalyzed deamination of cytosine to uracil.[3]
In the experiment, standard and extracted DNA(from hog thymus) and RNA (from yeast) samples were used for the hydrolysis study. Standards served as a basis whether the extracted DNA and RNA samples gave the expected results. For the base hydrolysis, 3 M NaOH was used, while 1.0 M HCl was used for acid hydrolysis. The test tubes were placed in boiling water for 1 hour. Heating hastens up the reaction, and an hour of boiling allots enough time for significant hydrolysis of the nucleic acids. Hydrol Hydrolysi ysis s studie studies s do not end on the hydrolys hydrolysis is react reaction ion alone. alone. It is also also necess necessary ary to do qualitative tests on the products of hydrolysis to be able to determine what species are present. In the
experiment, two common qualitative analysis were employed: the Bial/Orcinol’s test and the test for purine bases. Bial’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 7). The presence of furfural can be detected by the addition of a test reagent, known as the condensation reagent. The condensation reagent is generally generally a phenolic compound that reacts with 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.
Figure 7 . Formation of furfural form ribofuranose (cyclic form of ribose).
The other test perfor performe med d in the experim experiment ent is the test for the presence presence of purine purine bases. bases. Nucleotide bases are relatively insoluble in water. Depending on the pH, they may exist in two or more tautom tautomeri eric c forms forms (Figur (Figure e 8). 8). They They can be precip precipita itated ted by as their their silver silver salts by the addition addition of ammoniacal silver nitrate. A positive result is indicated by the presence of a white precipitate.
Figure 8. Tautomerism in nucleotide bases. In the experiment, after subjecting the nucleic acid samples to hydrolysis, qualitative tests were then performed. The results are summarized in Tables 1 and 2.
Table 1. RNA hydrolysis. Qualitative Acid Test Hydrolysat e Standard RNA
Bial’s Test Test for Purines
(+) (+)
Table 2 . DNA hydrolysis. Qualitative Acid Test Hydrolysat e Standard DNA
Bial’s Test Test for Purines
(-) (+)
Base Hydrolysat e Standard RNA
(+) (-)
Base Hydrolysat e Standard DNA (-) (-)
Acid Hydrolysat e Yeast RNA 1 2
Base Hydrolysat e Yeast RNA 1 2
(+) (+)
(+) (-)
(+) (+)
Acid Hydrolysat e DNA 1 2 (+) (+)
(-) (+)
(-) (-)
Base Hydrolysat e DNA 1 2 (-) (-)
(-) (-)
Unhydrolyze d Standard RNA
(+) (-)
Unhydrolyze d Standard DNA
(-) (+)
Unhydrolyze d Yeast RNA
1
2
(+) (-)
(+) (-)
Unhydrolyze d DNA 1
2
(-) (+)
(-) (+)
Table 1 and 2 summarizes the result of the qualitative tests performed. To simply the discussion it is better to divide the results into depending on the nucleic acid being considered. RNA Hydrolysis For the Bial’s test, all samples are expected to give positive results. Irregardless of whether the RNA is hydrolyzed or not, it has a ribose which can dehydrate to a furfural derivative. An anomaly is observed for the result of the base hydrolysate yeast RNA sample 2. For the test for purine bases, 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. Experimental results agree with the expected results. DNA Hydrolysis For the Bial’s test, all samples are expected to give a negative result. Though DNA has a ribose as a sugar component, it is a deoxyribose. Without a free hydroxyl group, it cannot further dehydrate to a furfural derivative that would give a positive result to the test. All o f the experimental results, except for the acid hydrolysate sample 2, agree with the expected results. For the test for purine bases, only those which underwent acid hydrolysis should give positive results. The principle behind is the same as explained in the RNA hydrolysis. Unhydrolyzed DNA samples gave erroneous results. A number number of samples samples gave erroneous results. results. There There is a possibili possibility ty that contaminatio contamination n could could have occurred in the course experiment leading to hydrolysis which should have not occurred, as in the case of the unhydrolyzed DNA samples. It is also possible that contaminants may react with test reagents to give false positive results. ` Only Only two two qua quali lita tati tive ve tes tests ts wer were e empl employ oyed ed in in the the expe experi rime ment nt.. Ther There e are are also also othe otherr meth method ods s by which DNA can be distinguished from RNA. One method involves the use of diphenylamine indicator. In this method, there is a need first for DNA to be acid-hydrolyzed at boiling temperature. The reaction requires the presence of a deoxyribose, therefore, specific for DNA. In these conditions, 2-deoxyribose is converted to ω-hydroxylevulinyl aldehyde, which then reacts with diphenylamine to yield a blue product.
Despite some errors incurred in the course of the experiment and in the results of the qualitative tests, the experiment was successful in fulfilling its objectives. Concept of nucleic acid hydrolysis has been successfully introduced and studied. The experiment was also able to show how qualitative tests are employed to characterize hydrolysates and be a gauge in the differentiation of RNA and DNA from one another. References [1] Matthews, C. K., et al. Biochemistry , 2nd ed.. Menlo Parks, Canada: Benjamin/Cummings Publishing Company, pp. 86-91. [2] Stryer, L., L., et. Al. Al. 2002.Biochemisry 2002.Biochemisry , 5th ed. New Yorkl: W. H. Freeman and Company, pp 117-119. [3] Adams, R., et al. The Biochemistry of Nucleic Acids. Acids. Google Google Books. Books. > Accessed 17 Aug 2008. [4] Mahato, R. I. Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids . Google Google Books. Books. > Accessed 17 Aug 2008. [5] “The Diphenylamine Diphenylamine Colorime Colorimetric tric Assay Assay and Method Method Validatio Validation”. n”. The Biotechnol Biotechnology ogy Project Project . < http://matcmadison.edu/biotech/resources/m http://matcmadison.edu/biotech/resources/methods/labManual/unit_4/ex ethods/labManual/unit_4/exercise_14.htm ercise_14.htm> > Accessed 18 Aug 2008.
