Circular Dichroism Spectra Of DNA Research Articles

DNA Complexes with Cobalt(II) Phthalocyanine Disodium Disulfonate.

The interaction of cobalt phthalocyanine disodium disulfonate (CoPc) with calf thymus DNA in solutions was investigated by UV/vis spectrophotometry, circular dichroism (CD), and hydrodynamic methods (viscosity and flow birefringence). Two types of CoPc binding to DNA were observed. Fast CoPc interactions with DNA via external binding to phosphates were accompanied by the formation of stack-type phthalocyanine structures on the periphery of the DNA helix. The optical absorption spectra of such CoPc complexes with DNA were analyzed in order to obtain a binding constant K = (4.8 ± 0.4) × 104 M–1. CD spectra show the increasing optical activity of phthalocyanines bonded to DNA. DNA plays the role of a matrix, contributing to an increase in their stacking interactions. The CD spectrum of DNA varies slightly. The second type of cobalt-to-DNA binding manifests itself over a certain time. It can be associated with the reorganization of ligands in the cobalt coordination sphere by introducing DNA atoms. In our experiments, such binding was observed after storage of solutions for approximately 20 h at a temperature of 4 °C. It was shown that the minor groove of DNA remains free in CoPc–DNA complexes. CoPc does not bind with the most important group for metal coordinating to DNA in the major groove (N7 guanine). We completely excluded the intercalation binding model. The planes of phthalocyanines in CoPc–DNA complexes are oriented predominantly normal to the axis of the DNA helix. DNA rigidity (persistent length) does not change. This follows from the data on the measurement of the optical anisotropy and intrinsic viscosity of DNA in complexes.

Complexation between DNA and Hydrophilic-Cationic Diblock Copolymers.

We examine connections among polycation composition, DNA-polycation binding thermodynamics, binding strength, and resulting complex properties, for circular and linear DNA and hydrophilic diblock copolymers possessing cationic blocks. Two poly(2-deoxy-2-methacrylamido glucopyranose)-block-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA), with block degrees of polymerization of PMAG56-b-PAEMA30 and PMAG52-b-PAEMA63, are employed. DNA binding behavior of these diblocks is also compared with that of a PAEMA homopolymer, in order to evaluate the role of the hydrophilic, charge-neutral PMAG block. In addition, DNA structure was varied, utilizing both circular and linear DNA with the same contour length. The enthalpy change due to DNA-polycation interactions (ΔHint) is observed via isothermal titration calorimetry (ITC) during titrations of DNA with the polycations. With circular DNA, a higher cationic content is found to result in a completion of binding with a smaller amount of polycation, as well as a larger initial ΔHint. In contrast to the common understanding that a neutral block simply provides colloidal stability, the PMAG block turns out to significantly impact both the extent of the binding and the size and dispersity of the final complexes. With a lower cationic content, the complex is less compact, but both the size and dispersity are more stable. Changes in circular dichroism spectra of DNA are shown to be correlated with PMAG-to-PAEMA block length ratio. PMAG52-b-PAEMA63 leads to stronger binding with DNA, compared to PMAG56-b-PAEMA30. Better-defined polyplexes and more disruption in the DNA helices are observed when the PMAG-to-PAEMA ratio is lower. All in all, while PMAG itself does not directly interact with DNA, the DNA-polycation binding turns out to be sensitive to the balance between the DNA-PAEMA attraction and PMAG solvation. In addition, it is confirmed that polyelectrolyte complexation is favored both entropically and enthalpically when the ionic strength of the solution is low. While only endothermic interactions occur in the buffered systems, exothermic initial interactions are observed in low-salt, unbuffered cases. Finally, complexes formed with linear DNA show clear bimodal size distributions, distinct from those formed with circular DNA. Collectively, these data provide insights into the controllable parameters in DNA-polycation complexation, which may advance the development of polymeric vehicles for large biomolecules such as nucleic acids.

Similarities and Differences between RNA and DNA Double-Helical Structures in Circular Dichroism Spectroscopy: A SAC-CI Study.

The helical structures of DNA and RNA are investigated experimentally using circular dichroism (CD) spectroscopy. The signs and the shapes of the CD spectra are much different between the right- and left-handed structures as well as between DNA and RNA. The main difference lies in the sign at around 295 nm of the CD spectra: it is positive for the right-handed B-DNA and the left-handed Z-RNA but is negative for the left-handed Z-DNA and the right-handed A-RNA. We calculated the SAC-CI CD spectra of DNA and RNA using the tetramer models, which include both hydrogen-bonding and stacking interactions that are important in both DNA and RNA. The SAC-CI results reproduced the features at around 295 nm of the experimental CD spectra of each DNA and RNA, and elucidated that the strong stacking interaction between the two base pairs is the origin of the negative peaks at 295 nm of the CD spectra for both DNA and RNA. On the basis of these facts, we discuss the similarities and differences between RNA and DNA double-helical structures in the CD spectroscopy based on the ChiraSac methodology.

Characterization of the binding of neomycin/paromomycin sulfate with DNA using acridine orange as fluorescence probe and molecular docking technique

The binding of neomycin sulfate (NS)/paromomycin sulfate (PS) with DNA was investigated by fluorescence quenching using acridine orange (AO) as a fluorescence probe. Fluorescence lifetime, FT-IR, circular dichroism (CD), relative viscosity, ionic strength, DNA melting temperature, and molecular docking were performed to explore the binding mechanism. The binding constant of NS/PS and DNA was 6.70 × 103/1.44 × 103 L mol−1 at 291 K. The values of ΔHθ, ΔSθ, and ΔGθ suggested that van der Waals force or hydrogen bond might be the main binding force between NS/PS and DNA. The results of Stern–Volmer plots and fluorescence lifetime measurements all revealed that NS/PS quenching the fluorescence of DNA–AO was static in nature. FT-IR indicated that the interaction between DNA and NS/PS did occur. The relative viscosity and melting temperature of DNA were almost unchanged when NS/PS was introduced to the solution. The fluorescence intensity of NS/PS–DNA–AO was decreased with the increase in the ionic strength. For CD spectra of DNA, the intensity of positive band at nearly 275 nm was decreased and that of negative band at nearly 245 nm was increased with the increase in the concentration of NS/PS. The binding constant of NS/PS with double-stranded DNA (dsDNA) was larger than that of NS/PS with single-stranded DNA (ssDNA). From these studies, the binding mode of NS/PS with DNA was evaluated to be groove binding. The results of molecular docking further indicated that NS/PS could enter into the minor groove in the A–T rich region of DNA.

Synthesis, characterization and DNA interaction studies of a new platinum(II) complex containing caffeine and histidine ligands using instrumental and computational methods

A new Pt(II) complex, [Pt(Caff)(His)(Cl)] (Caff is Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione) and His is l-Histidine), was synthesized and characterized using different physicochemical methods. The interaction of this complex with calf thymus DNA (ct-DNA) was investigated by absorption, emission, circular dichroism (CD), and viscosity measurements and molecular docking techniques. The calculated binding constant, Kb, was 5.3 × 103 M−1. In fluorimetric studies, the enthalpy and entropy of the reaction between the complex and ct-DNA showed that the reaction is exothermic (∆H = −184.07 kJ mol−1, ∆S = −551.97 J mol−1 K−1). CD spectra of DNA in the presence of different amounts of the complex showed little changes in both the negative and positive band intensities, which imply a non-intercalative mode between the DNA and the platinum complex. Furthermore, the study of molecular docking also indicated that the complex binds to DNA via a groove binding mode.

Helical Structure and Circular Dichroism Spectra of DNA: A Theoretical Study

The helical structure is experimentally determined by circular dichroism (CD) spectra. The sign and shape of the CD spectra are different between B-DNA with a right-handed double-helical structure and Z-DNA with a left-handed double-helical structure. In particular, the sign at around 295 nm in CD spectra is positive for B-DNA, which is opposite to that of Z-DNA. However, it is difficult to determine the helical structure from the UV absorption spectra. Three important factors that affect the CD spectra of DNA are (1) the conformation of dG monomer, (2) the hydrogen-bonding interaction between two helices, and (3) the stacking interaction between nucleic acid bases. We calculated the CD spectra of (1) the dG monomer at different conformations, (2) the composite of dG and dC monomers, (3) two dimer models that simulate separately the hydrogen-bonding interaction and the stacking interaction, and (4) the tetramer model that includes both hydrogen-bonding and stacking interactions simultaneously. The helical structure of DNA can be clarified by a comparison of the experimental and SAC-CI theoretical CD spectra of DNA and that the sign at around 295 nm of the CD spectra of Z-DNA reflects from the strong stacking interaction characteristic of its helical structure.

Spectroscopic Studies of DNA Interactions with Food Colorant Indigo Carmine with the Use of Ethidium Bromide as a Fluorescence Probe

The interaction of indigo carmine (IC) with calf thymus DNA in physiological buffer (pH 7.4), using ethidium bromide (EB) dye as a fluorescence probe, was investigated by ultraviolet-visible absorption, fluorescence, and circular dichroism (CD) spectroscopy, coupled with viscosity measurements and DNA-melting studies. Hypochromicity of the absorption spectra of IC and enhancement in fluorescence polarization of IC were observed with the addition of DNA. Moreover, the binding of IC to DNA was able to decrease iodide and single-stranded DNA (ssDNA) quenching effects, increase the melting temperature and relative viscosity of DNA, and induce the changes in CD spectra of DNA. All of the evidence indicated that IC interacted with DNA in the mode of intercalative binding. Furthermore, the three-way synchronous fluorescence spectra data obtained from the interaction between IC and DNA-EB were resolved by parallel factor analysis (PARAFAC), and the results provided simultaneously the concentration information and the pure spectra for the three reaction components (IC, EB, and DNA-EB) of the system at equilibrium. This PARAFAC demonstrated that the intercalation of IC molecules into DNA proceeded by substituting for EB in the DNA-EB complex. The calculated thermodynamic parameters, ΔH° and ΔS°, suggested that both hydrophobic interactions and hydrogen bonds played a predominant role in the binding of IC to DNA.

DNA Binding Studies of 3, 5, 6-Trichloro-2-Pyridinol Pesticide Metabolite

3, 5, 6-Trichloro-2-pyridinol (TCP) is a stable metabolite of two major pesticides, Chlopyrifos insecticide and Triclopyr herbicide, which are widely used in the world. The potential health hazard associated with TCP is identified due to its high affinity to the DNA molecule. Therefore, in this study, the interaction of native calf thymus DNA with TCP has been investigated using spectrophotometric, circular dichroism (CD), spectrofluorometric, viscometric and voltametric techniques. It was found that TCP molecules could interact with DNA via a groove-binding mode, as evidenced by hyperchromism, with no red shift in the UV absorption band of TCP, no changes in K(b) values in the presence of salt, no significant changes in the specific viscosity and CD spectra of DNA, and a decrease in peak currents with no shift in the voltamogram. In addition, TCP is able to release Hoechst 33258, a strong groove binder, in the DNA solutions. The results are indicative of the groove-binding mode of TCP to DNA.

DNA Binding Studies of Tartrazine Food Additive

The interaction of native calf thymus DNA with tartrazine in 10 mM Tris-HCl aqueous solution at neutral pH 7.4 was investigated. Tartrazine is a nitrous derivative and may cause allergic reactions, with a potential of toxicological risk. Also, tartrazine induces oxidative stress and DNA damage. Its DNA binding properties were studied by UV-vis and circular dichroism spectra, competitive binding with Hoechst 33258, and viscosity measurements. Tartrazine molecules bind to DNA via groove mode as illustrated by hyperchromism in the UV absorption band of tartrazine, decrease in Hoechst-DNA solution fluorescence, unchanged viscosity of DNA, and conformational changes such as conversion from B-like to C-like in the circular dichroism spectra of DNA. The binding constants (K(b)) of DNA with tartrazine were calculated at different temperatures. Enthalpy and entropy changes were calculated to be +37 and +213 kJ mol(-1), respectively, according to the Van't Hoff equation, which indicated that the reaction is predominantly entropically driven. Also, tartrazine does not cleave plasmid DNA. Tartrazine interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 3.75 × 10(4) M(-1).

Evidence of altered DNA integrity in the brain regions of suicidal victims of Bipolar Depression

Deoxyribonucleic acid (DNA) integrity plays a significant role in cell function. There are limited studies with regard to the role of DNA damage in bipolar affective disorder (BP). In the present study, we have assessed DNA integrity, conformation, and stability in the brain region of bipolar depression (BD) patients (n=10) compared to age-matched controls (n=8). Genomic DNA was isolated from 10 postmortem BD patients’ brain regions (frontal cortex, Pons, medulla, thalamus, cerebellum, hypothalamus, Parietal, temporal, occipital lobe, and hippocampus) and from the age-matched control subjects. DNA from the frontal cortex, pons, medulla, and thalamus showed significantly higher number of strand breaks in BD (P<0.01) compared to the age-matched controls. However, DNA from the hippocampus region was intact and did not show any strand breaks. The stability studies also indicated that the melting temperature and ethidium bromide binding pattern were altered in the DNA of BD patients’ brain regions, except in the hippocampus. The conformation studies showed B-A or secondary B-DNA conformation (instead of the normal B-DNA) in BD patients’ brain regions, with the exception of the hippocampus. The levels of redox metals such as Copper (Cu) and Iron (Fe) were significantly elevated in the brain regions of the sufferers of BD, while the Zinc (Zn) level was decreased. In the hippocampus, there was no change in the Fe or Cu levels, whereas, the Zn level was elevated. There was a clear correlation between Cu and Fe levels versus strand breaks in the brain regions of the BD. To date, as far as we are aware, this is a new comprehensive database on stability and conformations of DNA in different brain regions of patients affected with BD. The biological significance of these findings is discussed here.

Non‐covalent Interaction of Perfluorooctanoic Acid with DNA

AbstractHealth risk from exposure of perfluorochemicals to wildlife and human has been a subject of concern in many fields such as environmental ecology, toxicology, pathology and life sciences. The interactions of perfluorooctanoic acid (PFOA) with DNA were investigated by equilibrium dialysis, circular dichroism and isothermal titration calorimetry techniques under normal physiological conditions in vitro. The binding of PFOA to DNA was a non‐covalent interaction and corresponded to the Langmuir adsorption isotherm in a two‐step binding model, in which PFOA climbed along the backbones of DNA and then interacted with the homolateral bases via hydrophobic interactions. The saturation number of PFOA was calculated to be 0.64 per base‐pair of DNA. Such an interaction caused the enhancement of circular dichroism spectra of DNA at both 245 and 275 nm, indicating the change of DNA conformation. The acidic media, low electrolyte and temperature ≦35°C are comparatively more favorable for PFOA binding to DNA. This work provides a useful experimental strategy for studying the interactions of perfluorochemicals with biomacromolecules, aiming at a better understanding of the gene toxicity mechanism of perfluorochemicals.

DNA packaging in water solutions induced by polylysine and spermidine

The conformation properties of the DNA molecule complexed with polylysine (PLL) and spermidine (Spd) were studied by circular dichroism (CD), ultraviolet spectrophotometry, dynamic light scattering (DLS), viscosimetry, and dynamic birefringence. Experimental data suggest that when the concentration ratio of the monomer residues of polylysine and DNA (N/P) in moles is smaller than unity, the viscosity changes but slightly, whereas when N/P > 1, it abruptly drops to solvent viscosity. This is accompanied by changes in optical anisotropy and in the circular dichroism spectra of DNA. DLS data indicate that for N/P > 1, compact discrete particles are formed.

Structures, spectra, and DNA-binding properties of mixed ligand copper(II) complexes of iminodiacetic acid: The novel role of diimine co-ligands on DNA conformation and hydrolytic and oxidative double strand DNA cleavage

The coordination geometry around copper(II) in [Cu(imda)(phen)(H 2O)] ( 1) (H 2imda = iminodiacetic acid, phen = 1,10-phenanthroline) is described as distorted octahedral while those in [Cu(imda)(5,6-dmp)] ( 2) (5,6-dmp = 5,6-dimethyl-1,10-phenanthroline) and [Cu(imda)(dpq)] ( 3) (dpq = dipyrido-[3,2- d:2′,3′- f]-quinoxaline) as trigonal bipyramidal distorted square-based pyramidal with the imda anion facially coordinated to copper(II). Absorption spectral ( K b: 1, 0.60 ± 0.04 × 10 3; 2, 3.9 ± 0.3 × 10 3; 3, 1.7 ± 0.5 × 10 4 M −1) and thermal denaturation studies (Δ T m: 1, 5.70 ± 0.05; 2, 5.5 ± 10; 3, 10.6 ± 10 °C) and viscosity measurements indicate that 3 interacts with calf thymus DNA more strongly than 1 and 2. The relative viscosities of DNA bound to 1 and 3 increase while that of DNA bound to 2 decreases indicating formation of kinks or bends and/or conversion of B to A conformation as revealed by the decrease in intensity of the helicity band in the circular dichroism spectrum of DNA. While 1 and 3 are bound to DNA through partial intercalation, respectively, of phen ring and the extended planar ring of dpq with DNA base stack, the complex 2 is involved in groove binding. All the complexes show cleavage of pBR322 supercoiled DNA in the presence of ascorbic acid with the cleavage efficiency varying in the order 3 > 1 > 2. The highest oxidative DNA cleavage of dpq complex is ascribed to its highest Cu(II)/Cu(I) redox potential. Oxidative cleavage studies using distamycin reveal minor groove binding for the dpq complex but a major groove binding for the phen and 5,6-dmp complexes. Also, all the complexes show hydrolytic DNA cleavage activity in the absence of light or a reducing agent with cleavage efficiency varying in the order 1 > 3 > 2.

Structure of DNA Complexes with Nonhistone Chromosomal Protein HMGB1 in the Presence of Manganese Ions

The complexes of DNA–HMGB1 protein–manganese ions have been studied using the circular dichroism (CD) technique. It was shown that the interactions of both the protein and metal ions with DNA in this three-component system differ from those in two-component complexes. The manganese ions did not affect the CD spectrum of the free HMGB1 protein. However, Mn2+ ions induced considerable changes in the CD spectrum of free DNA in the spectral range of 260–290 nm. The presence of Mn2+ ions prevented the formation of the ordered supramolecular structures typical of HMGB1–DNA complexes. The interaction of manganese ions with DNA had a marked influence on the local DNA structure, changing the properties of protein-binding sites and resulting in a marked decrease in cooperativity of HMGB1–DNA binding. Such changes in the mode of protein–DNA interactions occurred at concentrations as small as 0.01 mM Mn2+. Moreover, the changes in local DNA structure induced by the manganese ions promoted the appearance of new HMGB1 binding sites in the DNA double helix. At the same time, interactions with the HMGB1 protein induced alterations in the structure of the DNA double helix, which increased with an increase in the protein-to-DNA ratio. These alterations made the DNA–protein complex especially sensitive to manganese ions. Under these conditions the Mn2+ ions strongly affected the DNA structure, which was reflected in abrupt changes in the CD spectra of DNA in the complex in the range of 260–290 nm. Thus, structural changes in the DNA double helix in three-component DNA–HMGB1–Mn2+ complexes result from the combined and interdependent interactions of DNA with Mn2+ ions and HMGB1 molecules.

Toroid formation in charge neutralized flexible or semi‐flexible biopolymers: potential pathway for assembly of DNA carriers

The theoretical state diagram for semi-flexible macromolecules such as DNA predicts that a tightly wound toroid can be a stable structure. Experimentally, toroids roughly 100 nm in diameter are routinely observed for DNA in the presence of multivalent cations at low DNA concentration. Theory also predicts toroids can form between non-DNA semi-flexible polymers and multivalent counterions. This phenomenon provides a means to co-package DNA with functionalized anionic polymers to create gene delivery systems. We show using electron microscopy that non-DNA polymers (polylysine, polyglutamic acid, and dextran sulfate) form toroids when mixed with multi- or polyvalent ions of opposite charge. The non-DNA toroids are similar in diameter to ones made with DNA. The results using dextran sulfate, a semi-flexible polymer, are explained by current theory. However, theory predicts that high flexibility in polypeptides should discourage their incorporation into stable toroids. To explain these latter observations we propose that charge neutralization facilitates secondary structure formation, which confers stiffness, thereby allowing stable toroids for the polypeptides studied. We measured the secondary structure of the toroid-forming polypeptides using circular dichroism (CD). The CD spectrum indicates the polypeptides undergo transitions from non-ordered structures (random coil) to ordered secondary structures (either alpha-helix or beta-sheet) upon charge neutralization which supports the hypothesis. The type of secondary structure is dependent on the type of multivalent counterion used to form the toroids. Formation of the polypeptide toroids confers resistance to heat denaturation of the resulting polypeptide secondary structure. The CD spectrum of DNA in a toroid also is changed from that of uncomplexed DNA, but all of the counterions used to form DNA toroids created structures with similar CD spectra in the DNA region (250-290 nm). The toroid structure obtained using DNA is observed in other semi-flexible non-DNA polymers such as dextran sulfate, and also in flexible polymers such as polylysine and polyglutamic acid upon charge neutralization with multivalent counterions. In the flexible polymers we propose that this phenomenon is due to induction of secondary structure upon charge neutralization, which decreases polymer flexibility, i.e. increases polymer stiffness, to enable toroid formation. These results have significant implications for the co-assembly of non-DNA anionic polymers with DNA to create nanoscopic gene carriers.

In situ measurement of circular dichroism of DNA adsorbing onto a solid surface

The adsorption process of DNA dissolved in aqueous solutions onto the surface of polyethyleneimine (PEI) has been examined by the observation of in situ circular dichroism (CD), including time-resolved measurements, to elucidate the conformation of DNA at the liquid/solid interface. The adsorption process can be characterized by two stages that are characterized in terms of CD. In the first stage, time ( t)<700 s, a slight change in the time-resolved CD spectra of DNA is observed, whereas the value of the induced CD of the dye intercalated in DNA is constant. This result can be explained by the interaction between DNA and PEI during the adsorption at the liquid/solid interface. The weakness of the interaction is attributed to the geometrical restriction of this interface. In the second stage, t>700 s, where no further adsorption occurs, a change in the induced CD as well as in the CD of DNA is observed. This change in the induced CD can be interpreted as a significant conformational change of DNA for stabilizing the ion complex with PEI.

Structure/Function Analysis of Peptoid/Lipitoid:DNA Complexes

Previous transfection studies of cationic peptoid polymers (N-substituted polyglycines) and cationic lipitoid polymers (peptoid–phospholipid conjugates) have shown that only the polymers which possessed a repeating (cationic, hydrophobic, hydrophobic) substituent sequence are efficient in gene transfer in vitro. To determine if there is a physical attribute of peptoid and lipitoid complexes that correlates with efficient gene transfection, biophysical, and transfection measurements were performed with polymer:DNA complexes containing each of seven structurally diverse peptoid polymers and two lipitoids that possess different hydrophobic substituents. These measurements revealed that the biophysical properties of these complexes (size, ζ-potential, ethidium bromide exclusion) varied with polymer structure and complex (+/−) charge ratio but were not directly predictive of transfection efficiency. Unique alterations in the circular dichroism spectra of DNA were observed in complexes containing several of the peptoids and both lipitoids, although FTIR spectroscopy demonstrated that the DNA remained in the B-form. The lack of correlations between the physical properties and the transfection activities of these polyplexes suggests that a further subpopulation examination of these complexes by these methods may reveal hidden structure–activity relationships. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association.

Biophysical Characterization of PEI/DNA Complexes

The main goal of this study was to determine the effects of polyethylenimine (PEI) molecular weight and structure (750 kDa, 25 kDa, 2 kDa branched, and 25 kDa linear PEI) and the nitrogen/phosphate (N/P) molar ratio on the physical properties and transfection efficiencies of PEI/DNA complexes. Fourier transform infrared spectroscopy revealed that DNA remained in the B conformation when complexed to all PEIs. Unique alterations in the circular dichroism spectra of DNA were observed in the presence of each PEI, whereas differential scanning calorimetry measurements showed that all PEIs examined destabilized supercoiled DNA at N/P < 3/1, but not at higher ratios. Isothermal titration calorimetry revealed the existence of protonation changes at low ionic strength due to possible shifts in pK(a) of the ionizable groups of PEI during complex formation. Twenty-five kilodalton branched and 25 kDa linear PEI complexes showed the highest transfection efficiencies at an N/P ratio of 6:1 in COS-7 and CHO-K1 cells, respectively. These investigations have detected alterations in the physical and colloidal properties of the complexes that were sensitive to polymer structure, molecular weight, and polymer/DNA ratio, but these properties did not directly correlate with their transfection efficiencies. To further probe any possible relationship between these parameters and activity, a more refined biophysical analysis of any subpopulations in these samples that may differ in transfection activity is suggested, although the existence of such species remains unknown.

Influence of copper(II) and magnesium(II) ions on the ciprofloxacin binding to DNA

The influence of magnesium(II) and copper(II) ions on the binding of ciprofloxacin to double stranded calf thymus DNA was studied by fluorescence emission spectroscopy, ultraviolet- and circular dichroism (CD) spectroscopy. The interaction of ciprofloxacin and copper(II) ions was followed by strong fluorescence quenching which was almost unaffected by the presence of DNA. On the other hand, only a slight decrease in fluorescence emission intensity, which was enhanced in the presence of DNA, was observed for ciprofloxacin interaction with magnesium(II) ions. Furthermore, magnesium(II) ions increase the thermal stability of the DNA, while, in the presence of ciprofloxacin, the degree of stabilisation is smaller. In contrast, copper(II) ions destabilise double helical DNA to heat, while ciprofloxacin slightly affects only the second transition of the biphasic melting curve of calf thymus DNA. Magnesium(II) ions at 25 °C induce conformational transitions of DNA at concentrations of 1.5 mM and 2.5 M, as monitored by CD. On the other hand copper(II) ions induce only one conformational transition, at a concentration of 12.7 μM. At higher concentrations of copper(II) ions ( c>700 μM) DNA starts to precipitate. Significant changes in the CD spectra of DNA were observed after addition of ciprofloxacin to a solution containing DNA and copper(II) ions, but not to DNA and magnesium(II) ions. Based on our spectroscopic results, we propose that copper(II) ions are not directly involved into ciprofloxacin binding to DNA via phosphate groups as it has been suggested for magnesium(II) ions.

WHEN POSITIVELY CHARGED MILK PROTEINS CAN BIND TO DNA

The binding of three esterified milk proteins (β-lactoglobutin, α-lactalbumin and β-casein) to plasmid DNAs at pH 7.1 was followed by agarose-gel electrophoresis. Highly esterified β-lactoglobulin and α-lactalbumin samples showed DNA-binding capacities comparable to those exhibited by native basic proteins such as lysozymes and histones. All the studied esterified β-casein samples failed to bind to DNA at the applied pH. Complete retardation of DNA migration on agarose gel was observed at a 1:1 ratio of protein basic groups (Lys + Arg) to DNA phosphate add groups in the case of highly esterified β-lactoglobulin, esterified α-lactalbumin and native basic proteins (lysozyme and histone). Binding capacity was dependent on the degree of esterification of the milk proteins. Hydrolysis of esterified milk proteins either suppressed or reduced their DNA-binding capacities according to the degree of hydrolysis and consequently to the average size of the resulting peptides. A prolonged peptic hydrolysis (25% degree of hydrolysis) completely suppressed DNA-binding capacity probably because of the small sizes of the resulting peptic peptides (< 1 kDa). Treatment with trypsin, which hydrolyzed the esterified proteins into relatively large peptide fragments, reduced the DNA-binding capacity to levels inversely proportional to the degree of hydrolysis. In the range of 2.7–12.3 kb, there was no influence of the DNA size on the binding of esterified milk proteins. The interactions DNA-esterified milk proteins did not depend on the DNA shape (circular or linear). Circular dichroism spectra of DNA in complex with methylated β-lactoglobulin were markedly altered as compared to those obtained when DNA was in complex with native β-lactoghbutin.