Knut Hildenbrand

Esr Spectra of Radicals of Single-Stranded and Double-Stranded DNA in Aqueous Solution. Implications for Oh-Induced Strand Breakage

In situ photolysis at 20 degrees C (argon plasma light source, lambda approximately greater than 200 mm) of oxygen-free solutions containing 2 mM H2O2 and heat-denatured, single-stranded (ss)DNA from calf-thymus resulted in the ESR spectra of the 6-hydroxy-5,6-dihydro-thymin-5-yl (1) and 5-methyleneuracil (3) radicals linked to the sugar-phosphate backbone. They were generated by reaction of OH radicals with DNA. By comparison of the decay characteristics of the ESR signals with rate constants from pulse-conductivity measurements [E. Bothe, G.A. Qureshi and D. Schulte-Frohlinde, Z. Naturforsch., 38c, 1030, (1983)] the thymine-derived radicals (1) and (3) can be excluded as precursors of the fast, dominating component of strand breakage of ssDNA. In the absence of H2O2 from native, double-stranded (ds)DNA an ESR signal was obtained (singlet, g approximately 2.004, delta v1/2 approximately 0.8 mT) which was assigned to the deprotonated guanine radical cation, [G.(-H)] of a DNA subunit. It is assumed that by the UV irradiation the guanine radical cation, (G+.), is generated, either by monophotonic photoionization or by electron transfer to pyrimidine bases. By rapid transfer of the bridging proton from (G+.) to the hydrogen bonded cytosine [G.(-H)] is formed. When photolysis of dsDNA was carried out in the presence of H2O2, reaction of photolytically generated .OH resulted in peroxyl radicals and purine radicals. The oxygen for formation of the peroxyl radicals is probably produced by reaction of [G.(-H)] with H2O2. Photolysis of N2O-saturated solutions containing dsDNA or ssDNA provided another possibility of generation of OH radicals. Under those conditions the .OH-induced radicals (1) and (3) were obtained not only from ssDNA but also from dsDNA.

Molecular events during the interaction of envelopes of myxo- and RNA-tumor viruses with cell membranes. A 270 MHz 1H nuclear magnetic resonance study

Abstract The nuclear magnetic resonance (NMR) spectra of chick embryo cells have been analyzed after exposure to Newcastle disease virus (NDV). Virions that contained the envelope glycoproteins in the cleaved form and, thus, had full biological activity have been compared to virions that had reduced infectivity due to the presence of uncleaved glycoprotein F. After exposure to infectious virus, drastic changes occurred in the signals assigned to choline and the hydrocarbon chains of fatty acids. These observations are interpreted to demonstrate alteration of the fluid lipid bilayer structure of the cell membranes. This is compatible with the concept of membrane fusion as a penetration mechanism for NDV. Virus containing uncleaved F glycoprotein did not alter the NMR spectra. This indicates that infection is blocked at the stage of penetration. Similar, though less pronounced, differences have been observed when the effects of highly infectious influenza virus containing the hemagglutinin in the cleaved form were compared to the effects of virus which had a lower infectivity due to the presence of uncleaved hemagglutinin. Thus, it appears that the hemagglutinin of influenza virus is involved in penetration and that cleavage is necessary for this function. Alterations of the NMR spectra of the membrane lipids have also been observed when susceptible chick embryo cells (C/E) were infected with Rous sarcoma virus of subgroup B. Such alterations did not occur when nonsusceptible cells (C/B) were used. Thus, infection appears to be blocked again at the stage of penetration.

Comparison of the reaction of ˙OH and of SO4–˙ radicals with pyrimidine nucleosides. An electron spin resonance study in aqueous solution

Reactions of photolytically generated ˙OH and SO4–˙ radicals with uridine, cytidine, 2′-deoxyuridine (dU) 2′-deoxycytidine (dC), and thymidine have been studied by e.s.r. spectroscopy under anoxic conditions. In the experiments with ˙OH, the spectra of the uracil compounds were dominated by the signals of radicals originating from ˙OH addition at the alkenic double bond of the nucleobase. No spectra were observed for the cytosine derivatives and thymidine. With SO4–˙, base radicals were generated from the deoxyribonucleosides [C(5)-OH-6-yl from dU, C(6)-OH-5-yl from thymidine, and a nitrogen-centred radical from dC] whereas the ribonucleosides lead to two different types of sugar radical. One of them is derived from the 2′-hydroxyalkyl radical by heterolytic elimination of the nucleobase and the other is the 3′-hydroxyalkyl radical which undergoes ring-opening by heterolytic cleavage of the C(4′)-oxygen bond at neutral and alkaline pH. Both the ˙OH and SO4–˙ radicals add to the base moieties in the primary step. The adduct radicals formed with ˙OH from uridine and dU are stable on the millisecond time-scale of the e.s.r. experiment whereas the sulphate adducts are too short-lived to be detected by e.s.r. In the deoxyribose derivatives they either hydrolyse (dU and thymidine) or eliminate SO42– and a proton (dC) whereas in the ribonucleosides they induce intramolecular H abstraction from positions 2′ and 3′ of the sugar residues.

N-NBD-l-α-Dilauroylphosphatidylethanolamine. A new fluorescent probe to study spontaneous lipid transfer

Migration of the fluorescent phospholipid N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-L-alpha-dilauroylphosphati dylethanolamine between small sonicated egg phosphatidylcholine vesicles was studied by use of the fluorescence resonance energy transfer method. Contrary to the results of lipid transfer experiments reported for acyl chain NBD-labeled phospholipids (Nichols, J.W. and Pagano, R.E. (1982) Biochemistry 21, 1720-1728), the migration kinetics of N-NBD-DLPE had to be described by a sum of two exponential functions. The fast component (t1/2 approximately equal to 38 min) was assigned to lipid transfer via soluble monomers and the slow component (t1/2 approximately equal to 400 min) to transbilayer motion. A reversible four-stage process is suggested as a kinetic model. Mathematical treatment of this scheme is given yielding an analytical expression for the time dependence of NBD emission intensity. The use of N-NBD-DLPE in the resonance energy transfer measurements offers the advantage of simple chemical synthesis of the fluorescent probe and leads to additional information on transbilayer motion which was not available with the NBD-labeled lipids used so far.

Membrane lipid dynamics and density dependent growth control in normal and transformed avian cells.

Abstract Membrane fluidity of normal chick embryo fibroblasts and normal Japanese quail fibroblasts and their Rous sarcoma virus and methylcholanthrene transformed counterparts was investigated using the technique of fluorescence depolarisation of 1,6-diphenylhexatriene incorporated in the whole cells and in their isolated plasma membrane vesicles. Normal cells and isolated plasma membranes of normal cells showed significant changes in fluidity as a function of population density while neither Rous sarcoma virus transformed nor methylcholanthrene tumor cells or their isolated plasma membrane showed this effect. Stimulation of growth by addition of calf serum to cultures of quiescent, density-inhibited normal cells was accompanied by rapid changes in the direction of increased membrane lipid fluidity. Neither sparse normal cells, nor sparse or dense transformed cells showed any significant fluidity change in their membrane lipids upon addition of serum. Enzyme and electron microscopic analysis of the ratios of different membrane types in each cell type showed that this ratio was invariant with respect to cell population density but different between transformed and normal cells. Hence, the fluidity changes observed, measured as the mean rotational correlation time of the fluorescene probe in the membrane lipids, truly reflect organisational differences, occurring as a function of population density in cultures of cells which retain density-dependent growth control.

Hydroxyl-radical-induced reactions of poly (acrylic acid); a pulse radiolysis, EPR and product study. Part II. Oxygenated aqueous solutions

Hydroxyl radicals were generated radiolytically in N2O–O2(4:1)-saturated dilute aqueous solutions of poly(acrylic acid), PAA. They react with PAA by abstracting H atoms from PAA in both the α- and βpositions to the carboxy group. The resulting PAA radicals are converted by O2 into the corresponding peroxyl radicals (k= 3.1 × 108 dm33 mol–1 s–1 at pH 3.5 and k= 1 × 108 dm3 mol–1 s–1 at pH 10, as measured by pulse radiolysis). The lowering of the rate constant at high pH has been attributed to a diffusion barrier for O2 exerted by the ion cloud around the charged macroradical. Although the lifetimes of the PAA peroxyl radicals (measured by EPR spectroscopy) increase with increasing pH, they still decay much faster than the very long-lived PAA radicals formed in the absence of O2. Concomitant with an increase in the lifetime of the peroxyl radicals, O2 uptake (in units of mol per mol PAA radicals initially formed) increases from 1.7 at pH 2.5 to 17 at pH 10 as does decarboxylation (from 1.0 at pH 3.5 to 13 at pH 10), scission (from 0.05 at pH 2 to 2.4 at pH 9) and the formation of acetylacetone-like products (from 0.35 at pH 3.5 to 1.05 at pH 10). The high yield of O2 uptake at pH 10 is mainly the result of repetitive abstraction of α-hydrogens by PAA α-peroxyl radicals. The acetylacetone-like products are formed by intramolecular 1,5-H-transfer reactions followed by a decomposition of the ensuing hydroperoxides. The chain breaks are formed in the course of the bimolecular termination reactions as has been shown by pulse radiolysis with low-angle laser light-scattering detection.

Radiolysis of poly(acrylic acid) in aqueous solution

Abstract Poly(acrylic acid), PAA, reacts with OH-radicals yielding -CHCH(CO 2 H)- (β-radicals) and -CH 2 C(CO 2 H)- (α-radicals) in a ratio of approximately 2:1. This estimate is based on pulse radiolysis data where the absorption spectrum of the PAA-radicals was compared with the spectra of α-radicals from model systems. The β-radicals convert slowly into α-radicals ( k = 0.7 s −1 at pH 10). This process has also been observed by ESR. At PAA-concentrations of 10 −2 mol dm −3 chain scission dominates over other competing reactions except at low pH. The rate of chain scission was followed by pulse conductometry and in the pH range 7–9 k = 4 × 10 −2 s −1 was observed. Oxygen reacts with PAA-radicals with k = 3.1 × 10 8 dm 3 mol −1 s −1 at pH 3.5 and k = 1.0 × 10 8 dm 3 mol −1 s −1 at pH 10. The corresponding peroxyl radicals undergo slow intramolecular H-transfer yielding a UV-absorbing product whose properties are that of 1,3-diketones.

Reaction of SO4–˙ with methylated uracils. An electron spin resonance study in aqueous solution

Radicals obtained by reaction of photolytically generated SO4–˙ with 1-methyluracil (1), 1,3-dimethyluracil (2), 1-methylthymine (3), 1,3-dimethylthymine (4), and 1,3,6-trimethyluracil (5) were studied by e.s.r. spectroscopy in aqueous solutions. In situ photolysis of neutral and acidic solutions containing (1) and persulphate in low concentrations (3mM) resulted in the e.s.r. spectrum of the C(5)-OH adduct radical. E.s.r. spectra obtained from (1) and (2) with high persulphate concentrations (30mM) were assigned to the C(6)-OH adduct radicals. It is proposed that this change in radical population is due to a persulphate-induced chain reaction which results in selective oxidation of the C(5)-OH radicals and simultaneous accumulation of the C(6)-OH adducts generated in side reactions. When the persulphate concentration was raised to 60mM, 5-oxo-6-yl radicals were formed in secondary processes from (1) and (2) besides the OH adducts. In contrast to these results the thymines (3) and (4) yielded only C(6)-OH adduct radicals. Addition of phosphate dianions to the photolysis solutions containing persulphate and the pyrimidine bases (1)–(4) resulted in the e.s.r. spectra of the C(6)-phosphate adduct radicals (pH 6.5–9.5). Identical spectra were obtained by reaction of (1)–(4) with HPO4–˙ radical anions generated by photolysis of Li4P2O8. The results of the experiments with 1,3,6-trimethyluracil (5) were completely different. First, reaction of SO4–˙ with (5), even at low persulphate concentration (3mM), did not lead to an OH adduct but to a 5-oxo-6-yl secondary radical. Secondly it was not possible to generate phosphate adduct radicals from (5) either with a mixture of persulphate and phosphate or with peroxodiphosphate. The spectral parameters of the radicals derived from (1)–(5) are given and possible pathways for the SO4–˙-induced radical formation are discussed.

Fluorescence studies of the incorporation of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled phosphatidylethanolamines into liposomes

Abstract The kinetics of interaction of aqueous suspensions of phosphatidylethanolamine derivatives with single bilayer egg yolk phosphatidylcholine (egg PC) vesicles has been studied. For this purpose 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) was reacted with the free amino group of l -α-phosphatidylethanolamine, -dilauroyl and -dimyristoyl to give N -NBD-DLPE (Ia) and N -NBD-DMPE (Ib). Electron microscopy and 31 P-NMR experiments performed with (Ia) indicated that the labeled lipids in aqueous buffer are present in the form of nonbilayer aggregates of 100–200 A diameter with a highly disordered headgroup arrangement. The time-evolution of the NBD emission intensity in the incubation mixtures of (I) with small and large unilamellar vesicles (SUV and LUV) could be fitted with a double exponential function. The fluorescence decay parameters of the NBD group changed during the reaction time of (I) with SUV but did not change when aggregates of (I) were mixed with LUV. A kinetic model was evaluated which assumes the adsorption of aggregates of (I) to the surface of the liposomes in a primary step. The relaxation characteristics of this heterogeneous lipid distribution towards a more homogeneous membrane structure can be explained by the presence of two non-fluorescent pools of (I) and a fluorescent one in the egg PC matrix. The rate constants for transfer of (I) between those kinetic pools as well as equilibrium emission intensities were found to decrease with increasing length of the fatty acid chains of (I). This behaviour was explained by differences in the properties of the clusters of (I) in the egg PC membranes.

Kinetics of calcium-induced mixing of lipids and aqueous contents of large unilamellar phosphatidylserine vesicles

Abstract Calcium ion-induced fusion events in suspensions of large unilamellar phosphatidylserine (PS) liposomes were monitored by fluorescence methods. Mixing of vesicle contents was studied by measuring the increase in terbium emission intensity due to formation of a complex between Tb 3+ ions and dipicolinic acid trapped in the liposomes. Lipid redistribution was determined with the aid of the resonance transfer of excitaton energy using dipalmitoylphosphatidylethanolamine labelled with the donor N -(7-nitro-2,1,3-benzoxadiazol-4-yl) or the acceptor tetramethylrhodamine at the free amino group. The two methods yielded significantly different results. While recombination of contents could not be detected at Ca 2+ concentrations below 2.5 mM the threshold concentration for lipid mixing was 1 mM. For saturating Ca 2+ concentrations (>5 mM Ca 2+ ) initial rates were higher by almost an order of magnitude for lipid mixing than for recombination of liposome contents. These observations indicate that the observation of rapid lipid mixing phenomena does not allow one to draw conclusions as to the fate of the enclosed volumes.