A flow EPR study of deformation and orientation characteristics of erythrocyte ghosts: A possible effect of an altered state of cytoskeletal network

Summary Using the flow EPR technique, we investigated the resealed ghost deformability in shear flow and the effects of the altered state of cytoskeletal network induced by hypotonic incubation of ghosts. Isotonically resealed ghosts in the presence of Mg-ATP, in which alteration of cytoskeletal network is not effected, have smooth biconcave discoid shapes, and show a flow orientation and deformation behavior similar to that of erythrocytes, except that higher viscosities are required to induce the same degrees of deformation and orientation as in erythrocytes. The flow behavior of resealed ghosts is Mg-ATP dependent, and the shape of the ghosts resealed without Mg-ATP is echinocytic. In contrast, the ghosts resealed by hypotonic incubation show a markedly reduced deformability even with Mg-ATP present. Nonreducing, nondenaturing polyacrylamide gel electrophoresis (PAGE) of the low ionic strength extracts from hypotonically resealed ghosts reveals a shift of the spectrin tetramer-dimer equilibrium toward the dimers. In the maleimide spin-labeled ghosts, the ratios of the weakly immobilized to the strongly immobilized EPR intensities are larger in hypotonically resealed ghosts than in the isotonically resealed ghosts, indicating an enhanced mobility in the spectrin structure in the former. Photomicrographs of hypotonically resealed ghosts show slightly stomatocytic transformations. These data suggest that the shape and the deformability loss in hypotonically resealed ghosts are related to an alteration of the spectrin tetramer-dimer equilibrium in the membrane. Thus, the shift of the equilibrium is likely to affect the regulation of the membrane deformability both in normal and pathological cells such as hereditary elliptocytes.     

Use of spin label and the flow-induced ESR spectral difference for studying erythrocyte deformation

ESR spin-labeling method is expanded to measure the macroscopic visco-elastic properties of erythrocytes. A suspension of erythrocytes with an incorporated fatty acid spin label was forced to flow through a flat ESR sample cells, and the ESR spectral change caused by the shear flow was utilized to assess the cell deformability. Chemical cross-linking or heat denaturation of membrane proteins to make the cells less deformable without any morphological change was found to reduce the relative spectral difference (delta h/h). This result indicates that the spectral difference is related to the cell deformation that accompanies the orientation of the cells in the shear flow. In addition, the average decay time (tau av) for the spectral difference observed when the flow was abruptly interrupted became shorter with an increase in the degree of cross-linking or heat-denaturation abruptly interrupted became shorter with an increase in the degree of cross-linking or heat-denaturation at 49 degrees C. Since the observed tau av is much shorter than the expected rotational correlation time for the erythrocyte, the decay is attributed to the deformation recovery process. It is demonstrated that the measurements of both delta h/h and tau av by ESR spectroscopy give qualitative information on the viscosity and the elasticity of the cell membrane system.     

A circular dichroism study of human erythrocyte ghost proteins during exposure to 2450 MHz microwave radiation

The effect of 2450 MHz microwave radiation on the proteins of human erythrocyte ghosts has been investigated using circular dichroism spectroscopy. A specially constructed waveguide inserted into the spectropolarimeter allowed the continuous recording of optical activity before, during and after microwave irradiation. The data indicate that high levels of microwave radiation (600 mW/g, specific absorption rate) induce decreases in α-helical conformation that may result from both thermal vibrations and increased strain on the intramolecular hydrogen bonds that maintain secondary structure. The latter effect may result from differential intramolecular interactions with the oscillating electric field. Spectrin (bands 1 and 2) isolated from the ghosts was more sensitive to microwave irradiation than intact ghosts, and spectrin-depleted vesicles were the least sensitive. The data, therefore, indicate that the α-helical conformation of spectrin is altered by high levels of microwave radiation.     

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

Virus-induced fusion of human erythrocyte ghosts I. Effects of macromolecules on the final stages of the fusion reaction

Human erythrocyte ghosts prepared by hypotonic hemolysis can be fused by Sendai virus, provided that certain macromolecules (bovine serum albumin, dextran and others) are sequestered in the ghosts. Since fusion of the ghosts is dependent on intactness of the F(fusion)-glycoprotein of the virion, and since the other requirements for this reaction are also similar to those for the Sendai virus-induced fusion of intact erythrocytes, this system can be used as a model for the Sendai virus-induced cell fusion reaction. Sequestered macromolecules seem to be required for rounding of locally fused ghosts. Under low osmotic swelling conditions, such as use of ghosts sealed without macromolecules or using bovine serum albumin-loaded ghosts sealed in the presence of external macromolecules, no apparently complete cell fusion (large spherical polyghost formation) could be observed. Even under these conditions, however, occurence of local cell fusion could be demonstrated either by transfer of fluorescent-labeled albumin from one ghost to an other, or by observation of polyghost formation after osmotic swelling in the cold. Thus, final stages of the fusion reaction can be divided into local cell-cell fusion which could not be observed by phase-contrast microscopy, and rounding (i.e. formation of spherical polyghost). For the observation of fusion of ghosts, the last step seems to be important.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Binding and incorporation of lecithin-cholesterol vesicles to lymphocytes: A spin-label study

Summary When lecithin-cholesterol vesicles, containing the membrane-bound spin probe 3-doxyl-cholestane, were set in contact with mouse lymphocytes, the vesicles adsorbed to the cell and vesicle-membrane components were transferred to it. The spin probe was enzymatically reduced at the inside of the cell membrane. The spin-label method provided a means to determine quantitatively the extent of vesicles adsorption and vesicle-cell fusion by measuring the transfer of vesicles membrane material to the cell. This method, together with the reduction of spin label by the cell, allowed also a quantitative estimate of the extent of endocytosis during cell-liposome interaction.     

Characterization of the Band 3 substrate site in human red cell ghosts by NDS-TEMPO,a disulfonatostilbene spin probe: The function of protons in NDS-TEMPO and substrate-anion binding in relation to anion transport

Summary NDS-TEMPO is a specific disulfonatostilbene spin label for the Band 3 substrate site (K. F. Schnell, W. Elbe, J. Käsbauer & E. Kaufmann,Biochim. Biophys. Acta 732:266–275, 1983). The pH dependence of NDS-TEMPO binding and of chloride and sulfate binding was studied in resealed human erythrocyte ghosts. pH was varied from 6.0 to 9.0. The ESR spectra from NDS-TEMPO-labeled red cell ghosts exhibited a strong immobilization of membrane-bound NDS-TEMPO. Changes of pH had no effect upon the mobility of membrane-bound NDS-TEMPO. A mutual competition between NDS-TEMPO binding and the binding of the substrate-anions, chloride and sulfate, was observed throughout the entire pH range. The maximal number of NDS-TEMPO binding sites per cell was in the range of 9.0×10⁵ to 1.10×10⁶ and was found to be insusceptible to changes of pH. The NDS-TEMPO/substrate-site and the chloride/substratesite dissociation constants amounted to 1.25 m and to 17mm and were independent of pH from pH 6.0 to 8.0, while the sulfate/substrate-site dissociation constant displayed a strong pH dependency with a maximum of 50mm at about pH 7.0. The NDS-TEMPO inhibition constants from the chloride and the sulfate flux experiments were 0.5 m (0°C) and 1.8 m (25°C), respectively, and are in close accordance with the NDS-TEMPO/substrate-site dissociation constants. Our studies provide strong evidence for the assumption that NDS-TEMPO binds in fact to the substrate site of Band 3. They show that the strong pH dependence of the chloride and of the sulfate transport cannot result from the pH dependency of substrate-anion binding, but point to the participation of ionizable regulator sites in transport catalysis. These regulator sites seem to be positioned outside the substrate site of the Band 3 transport domain.     

The sub-membrane reticulum of the human erythrocyte: A scanning electron microscope study

A web-like reticulum underlying the human erythrocyte membrane was studied at a resolution of 5–10 nm by means of a scanning electron microscope. The network was visualized in isolated membranes (ghosts) torn open to reveal their interior space and in residues derived from ghosts extracted with Triton X-100. It formed a continuous (rather than patchy) cover over the entire cytoplasmic surface, except where lifted off or torn away. Filaments (5–40 nm in diameter), annular figures (40–60 nm in diameter), and nodes (30–100 nm in diameter) were prominent in different networks. The dimensions of the filaments and the interstices in the reticulum varied with conditions, suggesting that the network has elastic properties. This reticulum is probably related to the erythrocyte membrane proteins spectrin and actin.     

Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH

Low temperature EPR spectroscopy was used to characterise Mycobacterium tuberculosis catalase-peroxidase in its resting ferric haem state. Several high spin ferric haem forms and no low spin forms were found in the enzyme samples frozen in methanol on dry ice. The EPR spectra depended not only on the pH but also on the buffer type. As a general trend, the higher the pH, the greater the ‘rhombic’ fraction of the high spin ferric haem that was observed. The rhombic form was characterised by well separated two lines in the g = 6 region whereas in the ‘axial’ form the two lines overlap. This pH dependence of the equilibrium of axial and rhombic ferric haem forms is also seen in rapidly freeze-quenched samples. Different high spin ferric haem forms were monitored during a 3 week storage of the enzyme at 4 °C. For some forms, extremal dependences, i.e. those progressing via maxima or minima over storage time, were found. This indicates that the mechanism of the time-dependent transition from one high spin ferric haem form to another must be more complex than a simple single site oxidation.     

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.     

The effect of ethanol on erythrocyte carbonic anhydrase isoenzymes activity: An in vitro and in vivo study

The ethanol is a widely consumed as sedative-hypnotic drug throughout the world. In this study, the effects of ethanol were investigated on carbonic anhydrase (CA) enzyme activities both in vitro in human erythrocyte and in vivo in Sprague-Dawley rat erythrocyte. For in vitro study, the human carbonic anhydrase-I (HCA-I) and -II (HCA-II) are purified by Sepharose 4B–L-tyrosine-sulphanilamide affinity chromatography. In vivo CA enzyme activity was determined colorimetrically by using CO₂-hydration method of Wilbur and Anderson. Rat blood samples were taken from each rat before and after the ethanol administration at different times (1 h, 3 h, and 5 h). Rat erythrocyte CA activity was significantly inhibited by pharmacological dosage of the ethanol (2 mL.kg^{? 1}) for up to 3 h (p < 0.001) following intraperitoneally administration. The ethanol showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity, determined by colorimetrically using the CO₂-hydratase method. The inhibitor concentrations causing up to 50% inhibition (IC₅₀) were 2.09 M for HCA-I (r²:0.9273) and 1.83 M for HCA-II (r²:9749). In conclusion, it was demonstrated that carbonic anhydrase enzyme in erythrocytes was significantly inhibited by the ethanol both in in vitro and in vivo.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Distribution of glycophorin on the surface of human erythrocyte membranes and its association with intramembrane particles: An immunochemical and freeze-fracture study of normal and En(a−) erythrocytes

Human erythrocyte membranes of the En(a–) blood group lack the major sialoglycoprotein (glycophorin). By absorption of a crude antiglycophorin antiserum with En(a–) membranes a specific antiglycophorin antiserum was obtained. By immune electron microscopy we showed that glycophorin is randomly distributed on the surface of normal erythrocytes. When polycationized ferritin, which mainly binds to glycophorin, was used as a marker a similar even labeling of normal erythrocyte membranes was seen. En(a–) membranes bound much less of this marker. In freeze-fracturing the intramembrane particles of both membrane types had a similar distribution and appeared in equal amounts. However, partial removal of spectrin from these membranes, followed by incubation at pH 6 resulted in more extensive aggregation of the particles in En(a–) membranes than in normal membranes. The results may be interpreted as glycophorin contributing by electrostatic repulsion to the random distribution of the intramembrane particles in normal cells. This repulsion is weakened in En(a–) cells by the lack of glycophorin.     

Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter Pi. In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W(ex) can be determined from Pi, and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < rho < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.     

Effects of phytohaemagglutinin, wheat-germ agglutinin, and concanavalin-A on the physical state of sialic acid and membrane proteins in human erythrocyte ghosts: a spin label study

Parallel experiments employing sialic acid- and protein specific spin labels have been performed to monitor the effects on the physical state of this carbohydrate and membrane proteins of human erythrocytes induced by the binding of three lectins, $\text{Phaseolus vulgaris}$ phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), and Concanavalin-A (Con-A). PHA and WGA, both of which are known to bind at different sites on the principal sialoglycoprotein of human erythrocytes, glycophorin, had markedly different effects: compared to control values, PHA decreased the apparent correlation time of the sialic acid specific spin probe by 10% while this parameter was decreased by 33% by WGA. The protein specific spin label also monitored differential effects of these lectins: the relevant electron spin resonance parameter (the W/S ratio) was reduced 33% by PHA and increased by WGA over 17% from that of control values. Con-A, which is known to bind to the principal transmembrane protein, Band 3, had no effect on sialic acid or membrane proteins as assessed by the two spin labels employed. These results suggest that (1) the effects of binding of these different lectins, two of which bind to the same cell surface receptor, can be discriminated by use of spin labeling methods; (2) binding events occuring at the cell surface have distinct and pronounced effects on the physical state of proteins within the membrane; (3) the different results with PHA and WGA both of which bind to glycophorin are indicative of multiple and complex interactions of this glycoprotein with the membrane proteins in the erythrocyte; and (4) that the spin labelling technique has the potential to investigate the relationships between cell-surface binding events to membrane structural-functional interactions.     

A new technique for the study of erythrocyte survival by double labeling and use of a well-type Ge detector

A double label procedure with⁵⁷Co and⁵⁸Co has been developed for detailed in vivo studies of erythrocyte survival. A well-type Ge detector is used in the measurements. The activities necessary for these experiments are very low, and the associated dose received by the test persons can be neglected.     

Comparative study on the properties of saturated phosphatidylethanolamine and phosphatidylcholine bilayers: Barrier characteristics and susceptibility to phospholipase A2 degradation

Comparative studies on bilayer systems of saturated phosphatidylcholines and phosphatidylethanolamines revealed a maximum in ionic permeability in phosphatidylcholine bilayers at the temperature of the gel to liquid-crystalline phase transition but such an increase in permeability was not detectable in bilayers of phosphatidylethanolamine. Furthermore, it was found that at the phase transition temperature the phosphatidylcholine bilayers are subject to rapid hydrolysis by pancreatic phospholipase A₂ whereas phosphatidylethanolamine bilayers are not. These differences are discussed in view of detailed information on the molecular organization in the gel and liquid crystalline phases of the two phospholipid classes.     

Spin label studies on rat liver and heart plasma membranes: Effects of temperature,calcium, and lanthanum on membrane fluidity

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(1 2,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(1 2,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19° and 28°C) and heart (between 21° and 32°C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl₂ to I(1 2,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37°C. Similarly, titrating I(1 2,3)-labeled heart plasma membranes with either CaCl₂ or LaCl₃ decreased the lipid fluidity at 37°C, although the magnitude of the La³⁺ effect was larger and occurred at lower concentrations than that induced by Ca²⁺; addition of 0.2 mM La³⁺ or 3.2 mM Ca²⁺ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe–probe interactions. Ca²⁺ and La³⁺ at these concentrations decrease the activities of such plasma membrane enzymes as Na⁺, K⁺-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

DNA-fiber EPR spectroscopy as a tool to study DNA-metal complex interactions: DNA binding of hydrated Cu(II) ions and Cu(II) complexes of amino acids and peptides

DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.