Janko N. Herak

Free radicals from single crystals of deoxyguanosine 5′-monophosphate (Na Salt) irradiated at low temperatures

SummaryIn single crystals of the DNA nucleotide 2′deoxyguanosine-5′phosphate (5′dGMP) X- orγ-irradiated at 4.2 K or 15 K, two primary radical species can be discriminated and assigned to the cation and anion of the guanine base,G(+) andG(−). Both species are unstable.G(−) partially transforms into a secondary radical at 4.2 K, the latter being the precursor to the dominant 300 K species formed by netH-addition to carbonC8. The secondary radical, together with another intermediate appearing at 77 K and perhaps connected with the anion decay could not be structurally identified. The guanine cationG(+) transforms upon annealing to temperatures above 77 K into a more stable species by deprotonation at positionN1.

An EPR study of the transfer and trapping of holes produced by radiation in guanine(thioguanine) hydrochloride single crystals.

Single crystals of guanine hydrochloride monohydrate, guanine hydrochloride dihydrate and anhydrous guanine dihydrochloride, doped with thioguanine, were irradiated with X and gamma rays. In all three systems the dominant radicals were associated with thioguanine. In the former two systems the stabilized species is the thiyl radical, formed by initial loss of an electron at some of the guanines in the crystal lattice, followed by hole migration to thioguanine and subsequent deprotonation of the radical formed. In the anhydrous guanine(thioguanine) dihydrochloride, that process is followed by acquisition of a chlorine ion. In the guanine hydrochloride monohydrate and guanine hydrochloride dihydrate lattices, systems of interacting closely spaced stacked bases and strings of chloride ions might support the migration of electrons and/or holes. In anhydrous guanine dihydrochloride, neither the bases nor the Cl- ions alone are capable of providing the means for the long-range electron, energy and spin transfer. It is the interchangeable sequence of the charged bases and the Cl- ions that makes the supporting strings or networks. The ultimate chlorination of the thioguanine-centered electron-loss radicals depends mainly on the availability of the Cl- ions and the space for their accommodation in the vicinity of the sulfur atom.

ESR evidence for the radiation-induced breakage of the sugar-phosphate bonds in nucleotides: single crystal of deoxycytidine 5'-monophosphate

Abstract Electron spin resonance has been used for the identification of the radicals in the gamma-irradiated single crystal of deoxycytidine 5′-monophosphate monohydrate. From the number and angular dependence of the proton couplings the =C(4′)—Ċ(5′)—H2(5′) radicals have been identified. These radicals are formed by the breakage of the sugar—phosphate bond.

ENDOR study of the stable radicals in γ- or X-ray-irradiated single crystal of deoxycytidine-5′-phosphate

Abstract The room-temperature stable radicals formed by γ- or X-ray-irradiation of deoxycytidine-5′-phosphate were studied by EPR and ENDOR spectroscopy. While EPR was of limited use because of the complexity of the spectra, the ENDOR spectroscopic analysis showed the presence of at least three types of radicals, two of which were analyzed in greater detail. One of the radicals is formed from the primary radical, analyzed earlier, by the decomposition of the furanose ring. The observed couplings correspond to the H (5′) , H′ (5′) , and H (4′) protons. In the second radical the unpaired electron is located mostly on the other side of the deoxyribose moiety, on C (1′) . The relatively isotropic hyperfine couplings of the two β protons and shown to correspond to H (2′) and H′ (2′) .

Postirradiation Long-Range Energy Transfer in a Single Crystal of Cytosine Monohydrate: An EPR Study

Thiocytosine molecules incorporated in the cytosine monohydrate crystal lattice act as traps for both electrons and holes. The radiation-induced cytosine ion radicals, C(+) and C(-), release their charge upon heating. The excess electrons and holes migrate long distances in the crystal lattice. The migration of holes has been demonstrated by the postirradiation, thermally activated accumulation of thiocytosine cation radicals, T(+), and the migration of electrons by formation of the S-centered radicals of an anionic nature. It is estimated that the migration length of the holes is at least 30 interbase distances, and the migration length of the electrons is more than 100 interbase distances. The selective formation of the cationic and anionic trap radicals, depending on the trap concentration, is discussed in terms of differences between the migration of electrons and holes.

Radiation energy transfer and trapping in single crystals of hemihydrate and hydrochloride of 5-methylcytosine doped with 5-methylthiocytosine : An EPR study

Abstract Two different crystals of 5-methylcytosine with 5-methylthiocytosine as impuritiy, irradiated with ionising radiation, exhibit quite different types of paramagnetic centres associated with thio impurities. In 5-methylcytosine hemihydrate there are two types of radicals with the unpaired spin located mainly on sulphur. Both are of thiyl structure, presumably derived by a loss of an electron from 5-methylthiocytosine. In doped 5-methylcytosine hydrochloride two distinct paramagnetic species with high spin density on chlorine have been detected. One of them has been spectroscopically characterised and assigned to an electron-deficient species with the unpaired electron shared by a sulphur and a chlorine atom, with some delocalization over the pyrimidine ring. Both crystal lattices of 5-methylcytosine support migration of electrons/holes. In the hemihydrate lattice, the base stacking interaction, although weak in comparison to that in DNA or some other molecular crystals of the nucleic-acid bases, might be responsible for that. In the hydrochloride lattice, the chlorine-ion strings or layers might provide the means for the hole transport. Both neutral and positively charged 5-methylcytosine is a good radiation energy trap (hole trap).

Conformational changes in yeast tRNATyr revealed by EPR spectra of spin-labelled N6-(Δ2-isopentenyl)adenosine residue

Temperature-induced conformational changes in the anticodon region of yeast tRNATyr were studied by EPR spectroscopy. The spin label 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl was attached to the N6-(delta2-isopentenyl)-adenosine residue in tRNATyr, previously made reactive by iodination. The labelled tRNATyr gave an asymmetrical triplet spectrum typical of rapidly tumbling nitroxide, with a rotational correlation time (tauc) of 0.65 ns. Spin-labelled tRNATyr was exposed to heating and cooling in three different buffers each with or without MgCl2. In each case the Arrhenius plot of --log tauc vs. inverse absolute temperature gave two straight lines, intersecting at a critical temperature (tcr). Above tcr, the anisotropy of the spectrum was not reduced and the activation energy of motion increased, indicating that the transition is associated with a conformational change of the macromolecule. Transitions in 0.05 M potassium phosphate (pH 8.0) and 0.02 M Tris - HC1 (pH 7.0) were observed at potassium phosphate (pH 8.0) and 0.02 M Tris - Hc1 (pH 7.0) were observed at approx. 37 degrees C. When 0.01 M mgCl2 was present in these buffers, transitions were shifted to 46 degrees and 53 degrees C, respectively. Transitions in 0.01 M sodium cacodylate were observed at temperatures which are significantly lower. Since all these transitions occur at temperatures considerably below those required to melt the helical regions of tRNA, and at least approximately 10 degrees C below those reported to break tertiary interactions, it is supposed that they reflect some reorientation of the anticodon region, e.g. a change in tilt of the bases.

Sigma radicals in gamma-irradiated single crystals of 2-thiothymine

Thioanalogs of the nucleic-acid bases are known photosensitive probes and good traps of radiation energy. Radiation-induced sulfur-centered free radicals stabilized on the base thioanalogs molecules are of the cationic origin. Their electronic configuration is of the π type, unless an electron-donating group, like Cl−, is added to the sulfur atom, forming the σσ* “ three-electron” S∴Cl bond. The σ radical observed in irradiated 2-thiothymine is formed simply by deprotonation of the pristine cation radical. The 2σ character of the radical is determined from the nature of the g tensor and the A(14N) coupling tensor and their relation to the radical skeleton. The deprotonation of the cation radical takes place at N(3) rather than at N(1), generally observed in the irradiated pyrimidine natural bases and their other thioanalogs.

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D(37) values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 x 10(5) and 1.4 x 10(5), were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 x 10(5)) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.

ESR spectroscopy of the sulphur-centered radicals derived from thiocytosine

Abstract Cytosine: thiocytosine crystals are found to be suitable for the observation of radiation-energy transfer to the sulphur-containing species. At low temperatures the thiocytosine cations are formed by a transfer of excitations to thiocytosine in the host matrix. At elevated temperatures the thiocytosine protonated anions are observed. The latter radicals are formed by an electron transfer from the cytosine anion in a thermally activated process, with a subsequent protonation on S-2. The principal elements of the g tensors ( g xx =2.132, g yy =2.004 and g zz =2.002 for the cation and g xx =2.066, g yy =2.008 and g zz =2.000 for the protonated anion) are in agreement with the g values for other sulphur-centered radicals of similar structures.