Copper(II)-DNA denaturation. I. Concentration dependence of melting temperature and terminal relaxation time

Both kinetic and equilibrium properties of DNA denaturation in the presence of copper(II) cation were studied by using optical techniques. Equilibrium properties of the reaction, measured in terms of T_m, the melting temperature, were shown to depend not on the overall but on the equilibrium concentrations of the species involved. Although T_m did increase with DNA concentration at low copper(II) concentrations, under similar conditions an increase in T_m with increasing copper(II) concentration was not observed. The kinetic properties of the reaction, characterized by the terminal relaxation time, were also found to depend on equilibrium concentrations of reactants. Application of standard methods of relaxation kinetics led to the proposal of a mechanism for copper(II)–DNA complex formation. That mechanism involves the rapid binding of two copper(II) ions to a reaction site on the polymer, followed by a slow, rate-limiting, first-order decay of the complex to the denatured state.     

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 degrees 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 microM. At higher concentrations of copper(II) ions (c>700 microM) 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.     

Studies of thermally induced denaturation of azurin and azurin derivatives by differential scanning calorimetry: evidence for copper selectivity

Azurin, a blue copper protein from Pseudomonas aeruginosa, and several derivatives of azurin have been studied by differential scanning calorimetry. Two well-separated, irreversible transitions are observed in a scan of apoazurin under a variety of conditions, and they are assigned to distinct steps in the denaturation process. No specific structural component can be assigned to the lower temperature transition, but a "flap" structure which is found near the metal binding site may be involved. Circular dichroic spectra suggest that melting of the beta-sheet structure, the main structural motif in the native protein, occurs during the second transition. With the exceptions of the Ni(II) and p-(hydroxymercuri)benzoate derivatives, the transitions are superposed in the metalated forms, and the enthalpies of denaturation are more endothermic. By comparison with other first-row divalent transition ions and especially Zn(II), the Cu(II) derivative exhibits the most endothermic denaturation process. Along with other data, this suggests that the binding energy is greater for Cu(II). It is postulated that the selectivity for copper over zinc arises because of the irregular binding geometry offered by the folded protein. Denaturation of the Hg(II) derivative is even more endothermic, confirming that the type 1 binding site has a very great affinity for Hg(II). Finally, when substoichiometric amounts of Hg(II) are added to the apoprotein, there is evidence that a novel mercury-bridged dimer of azurin forms.     

Effect of divalent copper ions on heat denaturation of DNA

Using the thermal denaturation method the effect of bivalent copper of (4-10(-6)-10(-3)) M concentrations on the helix-coil transition of DNA was studied in the solution of Na+ concentrations 10(-3)-10(-1) M. Unlike the previous studies, this paper makes allowance for the effect of impurity ions present in DNA and deionized water. It has been shown that in the region of low Cu2+ and Na+ concentrations, thermal stability increases, the melting range extends and the denaturation curves become asymmetric. At concentrations more than approximately 3-10(-5) M Cu2+, melting temperature starts to fall, and the range reduces to 1-1.5 degrees at [Cu2+] greater than or equal to 2-10(-4) M. As [Cu2+] reaches these values, the denaturation curve asymmetry and melting range increase again, which is due to the inversion of the relative stability of AT- and GC-pairs. Employing experimental and phase-transition-theory data for homopolymers, the constants of Cu2+ binding with phosphates and DNA bases were calculated. The concentration dependence of the DNA denaturation parameters was shown to be governed by the superposition of binding Cu2+ with phosphates and nucleic acid bases.     

Thermally induced denaturation and aggregation of BLG-A: effect of the Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> metal ions

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations ≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu²⁺ or Zn²⁺ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.     

Fine structures in denaturation curves of bacteriophage lambda DNA. Their relation to the intramolecular heterogeneity in base compositon.

Precise recording of polyphasic optical melting curves was carried out for three kinds of bacteriophage lambda DNA differing in length (lambdac1857s7, lambdacIb2 and lambdacIb2b5). Each of denaturation steps in melting profiles was characterized by two parameters, the melting temperature and the relative size. Any difference in fine structures in melting profiles was not recognized between the intact lambdacI857s7DNA and the DNA fragmented into halves. The change in fine structures in melting profiles caused by the deletions of the b2 and b5 region agreed qualitatively well with the prediction based on the physical and the genetical maps of phage lambda chromosome. The combined results indicate that, first, the well-known linear relationship between melting temperature and G+C content may apply also to each of denaturation steps in polyphasic melting curves due to heterogeneity of nucleotide distribution in a single DNA species, and, second, the effect of molecular ends on melting fine structures can be neglected at moderate salt concentration (0.01 M less than or equal to Na+ less than or equal to 0.2 M) for such a high molecular weight DNA. The heterogeneous distribution of nucleotides was derived for lambdaDNA and for its b2 and b5 regions.     

High-resolution thermal denaturation of DNA. I. Theoretical and practical considerations for the resolution of thermal subtransitions.

The fidelity achieved in first derivative profiles of DNA thermal denaturation is shown to depend on a number of factors including the thermal increment of data gathering, the precision of absorbance readings, and the manner in which data are smoothed prior to calculating the derivative of hyperchromicity. The closeness with which thermal denaturation data can be fitted by a cubic polynomial is carefully considered, and a derivation is presented for the estimated error in calculated values of the derivative of hyperchromicity with respect to temperature. After reviewing both theoretical and experimental evidence for the expected minimum width of a thermal transition in DNA, we conclude that thermal increments of 0.05°C or less are required for an adequate representation of transitions in naturally occurring DNA's. Data gathered under conditions meeting the requirements suggested here for quantitative recording of thermal denaturation profiles (Vizard and Ansevin, submitted for publication) show that virtually all of the high-resolution thermal denaturation profile of a simple, naturally occuring DNA may consist of small subtransitions, which we call thermalites. The finding of substransitions is consistent with current theories of DNA melting. A particularly well-resolved thermalite of λ bacteriophage DNA has a breadth of only 0.30°C (2σ width), and thus is narrower than previously reported thermal transitions for DNA. For this thermalite, the combination of width, shape, and position in the profile suggests that the substransitions observed in accurately recorded DNA thermal denaturation profiles are not described satisfactorily by existing theories. Knowledge of the requirements for the quantitative recording of thermal denaturation profiles should greatly favor the usefulness of denaturation experiments for physical genomic analysis.     

DNA interaction with Mn2+ ions at elevated temperatures: VCD evidence of DNA aggregation

Interaction of natural calf thymus DNA with Mn(2+) ions was studied at room temperature and at elevated temperatures in the range from 23 degrees C to 94 degrees C by means of IR absorption and vibrational circular dichroism (VCD) spectroscopy. The Mn(2+) concentration was varied between 0 and 1.3M (0 and 10 [Mn]/[P]). The secondary structure of DNA remained in the frame of the B-form family in the whole ion concentration range at room temperature. No significant DNA denaturation was revealed at room temperature even at the highest concentration of metal ions studied. However at elevated temperatures, DNA denaturation and a significant decrease of the melting temperature of DNA connected with a decrease of the stability of DNA induced by Mn(2+) ions occurred. VCD demonstrated sensitivity to DNA condensation and aggregation as well as an ability to distinguish between these two processes. No condensation or aggregation of DNA was observed at room temperature at any of the metal ion concentrations studied. DNA condensation was revealed in a very narrow range of experimental conditions at around 2.4 [Mn]/[P] and about 55 degrees C. DNA aggregation was observed in the presence of Mn(2+) ions at elevated temperatures during or after denaturation. VCD spectroscopy turned out to be useful for studying DNA condensation and aggregation due to its ability to distinguish between these two processes, and for providing information about DNA secondary structure in a condensed or aggregated state.     

Acid-base and copper (II) complexation equilibria of poly(inosinic)-poly(cytidylic)

The acid-base and copper (II) complexing properties of the heteropolynucleotide poly(inosinic)-poly(cytidylic) acid were studied by means of potentiometric, spectrophotometric, CD, and molecular fluorescence titrations in a working aqueous medium of 0.15 M ionic strength at 37°C. The study of the acid-base equilibria has proved the existence of four different species in the pH interval 2–11. Two new species are detected in the presence of copper(II) ion, at acidic and neutral pH values. Spectrophotometric, CD, melting, and fluorescence data were treated with a multivariate curve resolution procedure that allowed the determination of the number of species simultaneously present in each system [acid-base equilibria, copper(II) complexation equilibria], and the estimation of their concentration profiles and of the pure spectra for each species. From the concentration profiles an unambiguous assignment of which species are present at any pH conditions can be made. Furthermore, the knowledge of the pure uv-visible, fluorescence, and CD spectra recovered by the proposed resolution method for each species brings important structural information. When this mathematical approach was applied to the thermal denaturation data, the melting profile and the uv pure spectra for all the different species or conformations formed in the melting process, were calculated. © 1997 John Wiley & Sons, Inc. Biopoly 42: 271–283, 1997     

Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry

Thermal denaturation of nuclear DNA is studied in situ in individual cells or isolated cell nuclei by employing the property of the fluorochrome acridine orange (AO) to differentially stain native and denatured DNA and by using an automated flow-through cytofluorimeter for measurement of cell fluorescence. RNAse-treated cells, or cell nuclei, are heated, stained and measured while in suspension and AO-DNA interaction is studied under equilibrium conditions. Measurements are made rapidly (200 cells/sec); subpopulations of cells from a measured sample can be chosen on the basis of differences in their staining or light-scattering properties and analysed separately. DNA denaturation in situ is rapid; it approaches maximum during the first 5 min of cell heating. Divalent cations stabilize DNA against denaturation. At low pH the transition occurs at lower temperature and the width of the transition curves (‘melting profiles’) is increased. Decrease in ionic strength lowers the DNA melting temperature. This effect is much more pronounced in cells pretreated with acids under conditions known to remove histones. Histones thus appear to stabilize DNA in situ by providing counterions. At least four separate phases can be distinguished in melting profiles of DNA in situ; they are believed to indicate different melting points of DNA in complexes with particular histones. A decrease in cell (nuclear) ability to scatter light coincides with DNA melting in situ, possibly representing altered refractive and/or reflective properties of cell nuclei. Formaldehyde, commonly used to prevent DNA renaturation, is not used in the present method. The heat-induced alterations in nuclear chromatin are adequately stabilized after cell cooling in the absence of this agent. Cells heated at 60–85 °C exhibit increased total fluorescence after AO-staining, which is believed to be due to unmasking of new sites on DNA. This increase is neither correlated with DNA melting, nor with the presence of histones. Possibly, it reflects destruction of DNA superstructure maintained at lower temperatures by DNA associations with other than histone macromolecules (nuclear membrane).     

Denaturation of mouse satellite and ribosomal DNA during hydroxyapatite thermal chromatography of chromatin

Mouse DNA and chromatin were melted on hydroxyapatite and the denaturation profiles of ribosomal and satellite DNAs were followed by hybridization with their complementary RNAs. Neither ribosomal nor bulk DNA had significantly different melting profiles in chromatin as compared to DNA. However, most of satellite DNA eluted at higher temperature from chromatin than from purified DNA. One explanation for the higher melting temperature of mouse satellite DNA in chromatin suggests that the complex between this particular DNA component and at least some proteins in chromatin is more stable than the average DNA-protein interaction.     

Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers

The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins. Thermal melting profiles were measured for eight oligomers in 40 mM Tris acetate buffer. The observed melting temperatures of most oligomers correlated roughly with the mobilities observed at 20 degrees C; however, one oligomer was much more stable than the others. The melting temperatures of four of the oligomers were close to the values predicted by DINAMelt [Markham, N. R., and Zuker, M. (2005) Nucleic Acids Res. 33, W577-W581]; melting temperatures of the other oligomers differed significantly from the predicted values. Thermal melting profiles were also measured for two oligomers as a function of the Tris acetate buffer concentration. The salt concentration dependence of the melting temperatures suggests that 0.15 Tris+ ion per phosphate is released upon denaturation. Because the apparent number of Tris+ ions released is greater than that observed by others for the release of Na+ ions from similar hairpins, the results suggest that DNA hairpins (and, presumably, duplexes) bind more Tris+ ions than Na+ ions in solution.     

Electron microscopy of simian virus 40 DNA configuration under denaturation conditions.

After isolation, the DNA of simian virus 40 appeared as a negative supertwist (form I) or as an open circle with at least one single-strand scission (form II). Under the denaturation conditions usually applied, such as heating in the presence of formaldehyde or application of alkali, form I molecules could appear as "relaxed" circles without single-strand scissions (form I') containing denatured sites not visible under the electron microscope. Form II molecules, under these denaturation conditions, showed partial or complete strand separations allowing the construction of denaturation maps. By using a modified denaturation procedure, i.e., heating of isolated SV40 DNA in the presence of dimethyl sulfoxide and formaldehyde followed by keeping the DNA in this denaturation solution at room temperature for periods up to 3 weeks, partially denatured relaxed circles without single-strand scissions were produced (form I'D) in addition to completely denatured form II molecules. The absence of single-strand scissions in form I'D molecules was demonstrated by a second heat treatment, which did not change the configuration of this molecular form. Form I'D molecules, in contrast to form I', contained denatured sites clearly discerible under the electron microscope. This combined application of two subsequent denaturation steps (denaturation by heating followed by denaturation at room temperature and neutral pH) showed that the molecular configuration I'D originated in two steps. The heating procedure produced molecules not distinquishable by electron microscopy from form I. In contrast to form I, these molecules were assumed to possess "preformed" denaturation sites (form I). Further treatment of form I molecules with denaturation solution at room temperature finally transformed them into convalently closed, relaxed, partially denatured circles exhibiting strand separations easily measurable on electron micrographs (form I'D). Denaturation maps of form I'D molecules were constructed by computer and compared with denaturation maps derived from partially denatured form II molecules. From these denaturation maps it can be concluded that the melting of base pairs occurring during the transition of simian virus 40 DNA form I into form I'D also preferentially happened at sites rich in the bases adenosine and thymine.     

Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues

Two evolutionary lineages, called Trypanosoma cruzi I and II, have been identified in T. cruzi, the etiologic agent of human Chagas disease. Here, we describe a molecular strategy for direct genetic typing of these major groups of T. cruzi directly in human tissues. The protocol is based on heminested PCR amplification of the D7 region of the 24Salpha ribosomal DNA (rDNA), followed by identification of the products using denaturation curves in real time PCR. The repetitive nature of the gene, and the heminested PCR format insured the high sensitivity necessary to detect the presence of the very scarce T. cruzi DNA present in the chronically infected human tissues. There is 80% DNA sequence homology between the two 24Salpha rDNA alleles that define the T. cruzi I and II groups, sufficient to produce different thermal denaturation curves with melting temperature (TM) values of 81.7+/-0.43 and 78.2+/-0.33 degrees C (mean+/-SEM). Using this technical approach, we analysed tissue samples (esophagi, hearts and colon) from 25 different patients with the gastrointestinal or cardiac forms of Chagas disease; in all of them we found only the presence of T cruzi II. Previous epidemiological and immunological findings had already led to the idea that chronic human infections occurring in Brazil and Argentina might be primarily due to T. cruzi II strains, but all the evidence available had been indirect. Our findings provide definitive proof of this hypothesis and will also allow the establishment of which group of T. cruzi is responsible for Chagas disease in other countries.     

Intervention of glutathione in pre-mutagenic catechol-mediated DNA damage in the presence of copper(II) ions

The catechol-mediated DNA damage in the presence of Cu(II) ions involves oxidation of guanine to 8-oxoguanine (8-oxoG) and DNA strand scission. It proceeds through the reactive oxygen species (ROS) generation. The mutagenicity of 8-oxoG lesions is due to its miscoding propensity reflected in GC→TA transversion taking place during the DNA repair process. To gain new insights into the nature of catechol-mediated DNA damage and its prevention, we have investigated the changes in DNA melting characteristics and 8-oxoG formation as the indicators of DNA damage in a model calf-thymus DNA system. A novel fluorescence method for DNA melting temperature determination, based on DAPI fluorescent-probe staining, has been proposed. The DNA melting-onset temperature has been found to be more sensitive to DNA damage than the standard melting temperature due to the increased width of the melting transition observed in oxidatively damaged DNA. We have found that the efficiency of Fenton cascade in generating DNA-damaging ROS is higher for catechol than for GSH, two strong antioxidants, mainly due to the much longer distance between ROS-generating radical group in GS to nucleobases than that of semiquinone radical group to nucleobases (2.1nm vs. 0.27nm), making the ROS transport from GSH an order of magnitude less likely to damage DNA because of short lifetime of HO radicals. The antioxidant and DNA-protecting behaviors of GSH have been elucidated. We have found that the redox potential of GSH/GSSG couple is lower than that of catechol/semiquinone couple. Hence, GSH keeps catechol in the reduced state, thereby shutting down the initial step of the catechol-mediated Fenton cascade. The catechol-induced DNA damage in the presence of Cu(II) ions has also been confirmed in studies of ON-OFF hairpin-oligonucleotide beacons.     

Thermal denaturation profiles of deoxypolynucleotide-ligand complexes: semiempirical studies

We have semiempirically studied the thermal denaturation profiles of complexes formed between double strand polynucleotides and pure stabilizer nonspecific binding ligands. By using the McGhee model (J. D. McGhee, (1976) Biopolymers 15, 1345-1375) we have found a simple, analytical relationship between the melting temperature (Tm) and the Kh (intrinsic association constant), nh (apparent site size), and wh (cooperativity constant) values of the interaction. The validity of this approach strongly depends on the sigma value (sigma being the nucleation parameter of the DNA). Through the equation so obtained it is possible to calculate the Kh, nh, and wh values from the melting temperature of three experimental thermal denaturation profiles at different r (ligand/polynucleotide ratio) values. The method has been checked by studying the thermal denaturation profiles of daunomycin-poly(d(A-T)).poly(d(A-T)) complexes in two different salt concentrations. The results so obtained are compared with those previously described using other techniques. The applicability of the method here developed is discussed in relation with both the nature of the ligands and the value of the nucleation parameter (sigma).     

Studies on the thermal denaturation of nucleohistones

The thermal denaturation profiles of nucleohistone from calf thymus, sea urchin sperm and sea cucumber male gonad, are studied and compared under a variety of conditions. These include melting in the presence of either one of the following agents: urea, methanol, divalent cations or excess histones. The influence of ionic strength, pH, formaldehyde treatment and partial denaturation is also studied. Particular attention is given to the factors which influence the bimodal appearance of the profiles. The melting curves of the three materials used are qualitatively similar under all conditions, although they show quantitative differences. The histone:DNA ratio appears to be the most important parameter to define the denaturation properties of a given nucleohistone preparation. It is shown that redistribution of histones may determine the melting profile, since during denaturation histones can migrate from locally denatured regions towards those regions which contain native DNA. It is also shown that there are regions of phosphate negative charges of DNA not protected by histone. These regions can be protected against denaturation either by additional histones or by certain divalent cations. The results are interpreted in terms of the various models possible for the distribution of histones on DNA in native nucleohistone. Their biological significance is also discussed.     

Synthesis and copper-mediated nuclease activity of a tetracationic tris(2,2′-bipyridine) ligand

We report herein the synthesis of a novel tetracationic tris(2,2′-bipyridine) ligand 4. We show that this ligand metalated with copper(II), and in the presence of ascorbate as a reducing agent, strongly damages pUC18 plasmid DNA. Copper complex formation was demonstrated by ESI-MS (electrospray ionization-mass spectrum) at a 1:3 ligand to metal ratio. Binding of both 4 and its copper(II) complex to CT-DNA (calf thymus DNA) was characterized by viscosimetry, thermal denaturation and fluorescence-based competition assays. The viscosimetric data indicated that 4 and its copper(II) complex bind DNA through partial intercalation and thermal denaturation studies revealed a significant increase of duplex DNA stability in the presence of these species (ΔT_m = 16.4 and 18.3 °C, respectively). Moreover, 4 and its copper(II) complex were found to effectively compete with ethidium bromide for the intercalative binding sites of DNA. Overall, the copper(II)-4 complex constitutes a very efficient DNA cleaving agent in the presence of ascorbate. Experiments with scavengers further suggest that the generation of Cu(I), hydrogen peroxide, superoxide, hydroxyl radical and singlet oxygen-like species contributes to the DNA breakage induced by the Cu(II) complex of 4.     

Divalent metal ion effect on helix-coil transition of high molecular weight DNA in neutral and alkaline solutions

Effect of Mg(2+), Ca(2+), Ni(2+) and Cd(2+) ions on parameters of DNA helix-coil transition in sodium cacodylate (pH 6.5), Tris (pH 8.5) and sodium tetraborate (pH 9.0) buffers have been studied by differential UV-visible spectroscopy and by thermal denaturation. Anomalous behavior of the melting temperature T(m) and the melting interval ΔT in the presence of MgCl(2) was observed in Tris, but not in cacodylate or tetraborate buffers. Changes in the buffer type and pH did not influence T(m) and ΔT dependence on Ca(2+) and Cd(2+) concentrations. Decrease of the T(m) and ΔT of DNA in the presence of Ni(2+) and Cd(2+) was caused by preferential ion interaction with N7 of guanine. This type of interaction was also found for Mg(2+) in Tris buffer. The anomalous decrease in the T(m) and ΔT values was connected to formation of complexes between metal ions and Tris molecules. Transition of DNA single-stranded regions into a compact form with the effective radius of the particles of 300±100 ? was induced by Mg(2+) ions in Tris buffer.     

Further fluorospectrophotometric studies on the binding of acridine orange with DNA. Effects of thermal denaturation of DNA and additions of spermine, kanamycin, dihydrostreptomycin, methylene blue and chlorpromazine

Fluorospectrophotometric studies on the binding of acridine orange (AO) with calf thymus DNA showed that the thermal denaturation of DNA reduced markedly the fluorescence of Complex II and the extent of this decrease depended on the temperature to which the DNA solutions were heated. The denaturation was carried out in the absence and presence of AO (methods A and B, respectively), and then fluorescence measurements of solutions were carried out at 23 °C. The fluorescence intensity-heating temperature curves obtained by methods A and B were similar in shape to the usual melting curves of DNA and AO-DNA solutions, respectively. The higher midpoint value obtained with method B indicates the stabilizing activity of AO against denaturation. These findings support an intercalation model for Complex II and an external self-association binding model for Complex I.A high concentration of ethylene diamine (EDA) restored the fluorescence of denatured Complex II to about 80% of the intensity value of native Complex II. The effects of spermine, kanamycin and dihydrostreptomycin were much stronger than that of EDA.Methylene blue (MB) and chlorpromazine (CP) reduced the fluorescence of native Complex II markedly. Since the analysis of the difference absorption spectra declared that MB and CP were intercalated without release of bound AO, the interacting MB and CP were considered to weaken the interaction between AO and DNA bases, that made AO more fluorescent. Free radical (CP·) of CP was prepared by a new method using H₂O₂, peroxidase, and ascorbic acid. Intercalated CP· showed a much stronger quenching effect on Complex II, indicating that unpaired electron spin contained in the costacking unit between CP· and DNA bases might affect the fluorescence of the adjacent AO molecule by paramagnetic perturbation.