Deoxyribose determination

This is a spectrophotometric assay based on the reaction of diphenylamine with the deoxyribose moiety of DNA to produce a complex that absorbs at 600 nm. The reaction is specific for deoxyribose and RNA does not interfere. It can be used on relatively crude extracts where direct spectrophotometric determinations of DNA concentration are not possible. 

The quantitative determinations of the monosaccharides ribose and deoxyribose are given in Protocols 1.2.2 and 1.2.3, respectively. The following protocol is useful for all monosaccharides. 

An alternative method to investigate DNA strand breakage by OH radicals considers the surface accessibility of hydrogen atoms of the DNA backbone [102]. The solvent accessibility is 80% for the sugar-phosphates and —20% for the bases. This method allows a more direct determination of reaction of OH radicals with the individual deoxyribose hydrogens [103,104]. Recent studies show trends in reactivity of OH radicals closely follow the accessibility of the solvent to various deoxyribose hydrogens [105,106]. 

What s DNA Deoxyribonucleic acid, the helical ladderlike chain of molecules that makes up genes. DNA consists of a sugar molecule called deoxyribose (it is somewhat related to glucose), a nitrogen-containing molecule called a base, and phosphate atoms bonded to the other two components. It is the sequence of base pairs (one base on each strand) in DNA that determines the end-product (e.g., protein). The human genome— the entire DNA content of a human being—contains approximately 3 billion base pairs. 

Steenken et al. have concluded that in double-stranded DNA direct hydrogen atom abstraction from 2 -deoxyribose by G(-H) radical is very unlikely due to steric hindrance effects and a small thermodynamic driving force [94]. The EPR studies performed in neutral aqueous solutions at room temperature have shown that, in the absence of specific reactive molecules, the lifetime of the G(-H) radical in double-stranded DNA is as long as -5 s [80]. Therefore, the fates of G(-H) radicals are mostly determined by the presence of other reactive species and radicals. Thus, the G(-H) radical can be a key precursor of diverse guanine lesions in DNA. In the next section we begin from a discussion of the site-selective generation of the G(-H) radical in DNA, and then continue with a discussion of the reaction pathways of this guanine radical. 

Roboz J, Suttajit M, Bekesi JG (1981) Elimination of 2-deoxyribose interference in the thiobarbituric acid determination of N-acetylneuraminic acid in tumor cells by pH-dependent extraction with cyclohexanone. Anal Biochem 110 380-388... 

The rate constants for the reactions between OH and a range of ethers and hydroxy ethers have been reported at 298 K233 as well as those for reactions between dimethyl ether and methyl f-butyl ether over the range 295-750 K.234 Data from the former study show deviations from simple structure-activity relationships which were postulated to arise due to H-bonding in the reaction transition states.233 The atmospheric lifetime of methyl ethyl ether has been determined to be approximately 2 days.235 Theoretical studies on the H-abstraction from propan-2-ol (a model for deoxyribose) by OH have been reported using ab initio methods (MP2/6-31G ).236 The temperature dependence (233-272 K) of the rate coefficients for the reaction of OH with methyl, ethyl, n-propyl, n-butyl, and f-butyl formate has been measured and structure-activity... 

Ribose and deoxyribose contribute to the formation of RNA and DNA, respectively. Binding of a base to the pentose yields a nucleoside, and binding of a phosphoric acid to the nucleoside forms a nucleotide. Finally, different nucleotides bound together form a nucleic acid. There is a specific complementary nature to the bases adenine with thymine and guanine with cytosine. The C-G pair has three hydrogen bonds, while the A-T pair has only two, thus preventing incorrect pairing. The sequence of the bases determines the primary structure of the DNA. 

So far we have seen that ionization creates a hole and ejects and electron. In DNA the electron is captured exclusively by the pyrimidine bases while the holes are distributed between guanine and the deoxyribose. The next problem to solve is to determine the free radical yield in DNA and to correlate this yield with the yield of strand breaks. These are very challenging experiments since there are so many factors influencing radicals yield. 

Bernhard and co-workers have performed a series of experiments to determine the mechanisms of DNA strand breakage by direct ionization of plasmid DNA. A big surprise in this work was the discovery that the total yield of single strand breaks exceeds the yield of trapped sugar radicals. Even at very low hydration levels (2.5 waters per nucleotide residue) nearly 2/3 of the strand breaks are derived from precursors other than deoxyribose radicals [74], The authors conclude that a majority of the strand breaks observed do not result from dissociative electron capture, homolytic bond cleavage from excited states, or from hydroxyl radical attack. Rather, the authors conclude that doubly oxidized deoxyribose is responsible for the high yield of strand breaks. 

As described in the previous section, experiments on LEE induced desorption of H-, O- and OH- from physisorbed DNA films, made it possible to demonstrate that the DEA mechanism is involved in the bond breaking process responsible for SB. The abundant H yield was assigned to the dissociation of temporary anions formed by the capture of the incident electron by the deoxyribose and/or the bases, whereas O production arose from temporary electron localization on the phosphate group [47], However, the source of OH- could not be determined unambiguously, and Pan et al. suggested that reactive scattering of O- may be involved in the release of OH [47], To resolve this problem, Pan and Sanche [58] investigated ESD of anions from SAM films of DNA. Their measurements allowed both the mechanism and site of OH- production to be determined. 

The degradation of deoxyribose is determined by a method akin to the conventional thiobarbituric acid assay for aldehydic products (see Chapter 5 for more information). 

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