Studies on poly(L-lysine50, L-tyrosine50)-DNA interaction

Interaction between poly(Lys⁵⁰, Tyr⁵⁰) and DNA has been studied by absorption, circular dichroism (CD), and fluorescence spectroscopy and thermal denaturation in 0.001M Tris, pH 6.8. The binding of this copolypeptide to DNA results in an absorbance enhancement and fluorescence quenching on tyrosine. There is also an increase in the tyrosine CD at 230 nm. The CD of DNA above 250 nm is slightly shifted to the longer wavelength which is qualitatively similar to, but quantitatively much smaller than, that induced by polylysine binding. At physiological pH the poly(Lys⁵⁰, Tyr⁵⁰)–DNA complex is soluble until there is one lysine and one tyrosine per nucleotide in the complex. The same ratio of amino acid residues to nucleotide has also been observed in copolypeptide-bound regions of the complex. The addition of more poly(Lys⁵⁰, Tyr⁵⁰) to DNA yields a constant melting temperature, T_m′, for bound base pairs at 90°C which is close to that of polylysine-bound DNA under the same condition. The melting temperature, T_m, of free base pairs at about 60°C on the other hand, is increased by 10°C as more copolypeptide is bound to DNA. As the temperature is raised, both absorption and CD spectra of the complexes with high coverage are changed, suggesting structural alteration, perhaps deprotonation, on bound tyrosine. The results in this report also suggest that intercalation of tyrosine in DNA is unlikely to be the mode of binding.     

Spectroscopic and equilibrium dialysis studies of the binding of acridine and 10-methylacridine to DNA in the pH range 4–10

For a better understanding of the interactions between DNA and various acridine dyes, the binding of acridine (Acr) and 10-methylacridine (MeAcr) to native and heat-denatured calf-thymus DNA was studied in the pH range between 4 and 10 by the equilibrium dialysis and spectroscopic methods. The binding between DNA and the dyes was predominantly electrostatic. The amount of bound Acr varied with pH, mixing ratio (P/D), and the DNA conformation, and reached a maximum at pH = 5.2. The amount of bound MeAcr was constant in the pH range 5–9. The apparent binding constants of these dyes were obtained at some pH, and they were found to vary with P/D for native DNA-dye complexes. The pure spectra of bound Acr and MeAcr could be unmasked. The bound Spectra were bathochromic and hypochromic relative to the spectra of free days. Acridine bound to native DNA was shown to undergo structural changes from an acridiniumlike to a neutral acridinelike form as the pH of solutions was varied. The pK value for the transition between the bound forms was evaluated to be 7.3. The extrinsic Cotton effects of the bound dyes were observed in the DNA-Acr and-MeAcr complexes and varied with pH and the conformation of DNA.     

Melting and premelting phenomenon in DNA by laser Raman scattering.

Raman spectra of DNA from calf thymus DNA have been taken over a wide range of temperatures (25°–95°) in both D₂O and H₂O. A study of the temperature dependence of the Raman spectra shows that the temperature profiles of the intensities and frequencies of the various bands fall into four different categories: (1) base bands that show a reversible increase in intensity prior to the melting region, i.e., a definite premelting phenomenon; (2) base bands that show little or no temperature dependence; (3) deoxyribose-phosphate backbone vibrations that show no temperature dependence up to the melting region, at which point large decreases in intensity occur; and (4) slow frequency changes in certain in-plane vibrations of guanine and adenine due to deuteration of the C-8 hydrogen of these purines in D₂O. Certain Raman bands arising from each of the four bases, adenine, thymine, guanine, and cytosine have been found to undergo a gradual increase in intensity prior to the melting region at which point large, abrupt increases in intensity occur. The carbonyl stretching band of thymine, involved in the interbase hydrogen bonding actually undergoes both a gradual shift to a lower frequency as well as an increase in intensity. These changes provide evidence that some change in the geometry of the bases relative to each other begins to occur around 50°C, well below the melting region of 70°–85°C. From the spectra taken at various temperatures, the DNA appears to remain in the B conformation until the melting point is reached, at which time the DNA progresses into a disordered random-coil form. No A-form conformation is found either in the premelting or the melting region.     

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.     

Thermodynamics of mucopolysaccharide–dye binding. II. Binding constant and cooperativity parameters of acridine orange–dermatan sulfate system

In the acridine orange–dermatan sulfate system, free and bound dye can be distinguished from each other spectroscopically. This permits the use of fluorometric methods to study the binding of acridine orange to the acid mucopolysaccharide dermatan sulfate. Experiments were conducted at 24°C in 10^?3 M citrate/phosphate buffer at pH = 7.0. The binding of the dye is highly cooperative, as evidenced by considerable interaction between adjacent bound dye molecules. Analysis of the data indicates that dermatan sulfate binds 2.3 ± 0.3 mol of acridine orange per dermatan sulfate uronic acid residue with a cooperative binding constant, K_q ranging from 4.9 to 6.0 × 10⁵ M^?1 which corresponds to a free energy of 7.74 ? ΔG° ? 7.86. The cooperativity parameter q apparently increases with increasing polymer-to-dye ratio.     

Discrimination by temperature of opiate agonist and antagonist receptor binding.

Variations in incubation temperature can markedly differentiate opiate receptor binding of agonists and antagonists. In the presence of sodium increasing incubation temperatures from 0° to 30° reduces receptor binding of ³H-naloxone by 50% while tripling the binding of the agonist ³H-dihydromorphine. Lowering incubation temperature from 25° to 0° reduces the potency of morphine in inhibiting ³H-naloxone binding by 9-fold while not affecting the potency of the antagonist nalorphine. At temperatures of 25° and higher the number of binding sites for opiate antagonists is increased by sodium and the number of sites for agonists is decreased by sodium with no changes in affinity. By contrast, in the presence of sodium lowering of incubation temperature to 0° increases opiate receptor binding of the antagonist naloxone by enhancing its affinity for binding sites even though the total number of binding sites are not changed.     

Investigation of DNA structural changes by infrared spectroscopy

DNA-oriented samples of various origins were studied under different conditions of humidiity and sodium chloride content by means of infrared spectroscopy. (1) Oriented DNA (M. Lysodeikticus, E. coli, calf thymus and salmon sperm) films at 3–4% sodium chloride yield polarized spectra which show drastic changes at relative humidities (r.h.) between 94% and 0% indicative of conformational changes: B form → a form → disordered form The measurements of the infrared dichroism at frequencies of about 1230 cm^?1 and at about 1090 cm^?1 allow one to determine the orientation of the phosphate group, whereas the measurements at 1710 cm^?1 characterize the base orientation. At humidities higher than 90% r.h. (B form) the bisector of OPO forms an angle of 70° relative to the helix axis, whereas at lower humidities, between 75% and 50% r.h. (A form) a rotation to about 45° is observed. Simultaneously, the 0—0 line of phosphate group changes its orientation from 55° to 65° to the helix when B → A transition takes place. The results are in general agreement with that of X-ray diffraction and allow one to determine the orientation of the phosphate group with greater precision. (2) The B–A conformational change is not observed for satellite DNA, isolated from Cancer pagurus, of which the guanine + cytosine content is below 5%. As a function of decreasing humidities, one observes the transition: B form → disordered form A diagram of conformational changes of DNA's as a function of base composition and of r.h., suggests that B–A transition will occur for DNA of relatively higher G + C content, whereas for high (A + T) content, base sequence may be of importance. The B–A transition is prevented in DNA at a relatively high or very low sodium chloride content.     

Determining the interaction behavior of calf thymus DNA with berberine hydrochloride in the presence of linker histone: a biophysical study

Abstract

The binding of small molecules with histone-DNA complexes can cause an interference in vital cellular processes such as cell division and the growth of cancerous cells that results in apoptosis. It is significant to study the interaction of small molecules with histone-DNA complex for the purpose of better understanding their mechanism of action, as well as designing novel and more effective drug compounds. The fluorescence quenching of ct-DNA upon interaction with Berberine has determined the binding of Berberine to ct-DNA with K_sv?=?9.46?×?10⁷ M^?1. K_sv value of ct-DNA-Berberine in the presence of H1 has been observed to be 3.10?×?10⁷ M^?1, indicating that the H1 has caused a reduction in the binding affinity of Berberine to ct-DNA. In the competitive emission spectrum, ethidium bromide (EB) and acridine orange (AO) have been examined as intercalators through the addition of Berberine to ct-DNA complexes, which includes ctDNA-EB and ctDNA-AO. Although in the presence of histone H1 , we have observed signs of competition through the induced changes within the emission spectra, yet there has been apparently no competition between the ligands and probes. The viscosity results have confirmed the different behaviors of interaction between ctDNA and Berberine throughout the binary and ternary systems. We have figured out the IC50 and viability percent values at three different time durations of interaction between Berberine and MCF7 cell line. The molecular experiments have been completed by achieving the results of MTT assay, which have been confirmed to be in good agreement with molecular modeling studies.

Communicated by Ramaswamy H. Sarma     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Transcriptional activity and chromatin structural changes in a temperature-sensitive mutant of BHK cells blocked in early G1.

AF8, a temperature-sensitive mutant of BHK $\text{21}\text{13}$ cells, has been shown to arrest at the non-permissive temperature in the G1 phase of the cell cycle. When AF8 cells are released from density-dependent arrest of growth by trypsinization and replating at lower density, 60–80% of the cells enter DNA synthesis and divide at the permissive temperature (33 °C), while only 20% enter DNA synthesis at the non-permissive temperature (39.5 °C). The temperature-sensitive block has been localized 4 to 8 h before the onset of DNA synthesis which begins at 12 h after stimulation. Two biochemical events of the prereplicative phase have been temporally related to this temperature-sensitive block. RNA synthesis as measured in isolated nuclei increases initially at both temperatures, then levels off and declines to control levels at 39.5 °C while continuing to increase at 33 °C. Parallel changes are found in circular dichroism spectra and ethidium bromide binding capacity of isolated chromatin. The results suggest that these biochemical changes are involved in the regulation of the prereplicative phase and the subsequent entrance of cells into DNA synthesis.     

Benzodiazepine receptors: temperature dependence of [3H]flunitrazepam binding.

The characteristics of [³H]flunitrazepam binding to brain specific benzodiazepine receptors were determined at varying temperatures. The rates at which [³H]flunitrazepam associated with and dissociated from benzodiazepine receptors increased with increasing temperatures. The dissociation constant (K_D) also increased with increases in temperature. The (K_D) determined by Scatchard analyses of saturation isotherms showed a similar change with changes in temperature. The maximal binding capacity (Bmax) did not change with changes in temperature. The inhibitory constants of several benzodiazepines to inhibit [³H]flunitrazepam binding to brain were also higher at 37°C than at 0°C, suggesting that the binding affinity of all benzodiazepines to brain benzodiazepine receptors is lower at 37°C than at 0°C. Van't Hoff analysis of [³H]flunitrazepam binding to brain at different temperatures reveals two linear components to this relationship.     

Effect of temperature on dose-dependent changes in sedimentation characteristics of bacterial DNA produced, in vivo, by near-ultraviolet irradiation and 8-methoxypsoralen

Dose-dependent changes in the sedimentation characteristics of bacterial DNA are produced, in vivo, by near-ultraviolet irradiation in the presence of the photosensitizer, 8-methoxypsoralen. These changes are probably due to DNA cross links and are associated with both lethality and mutation induction in bacteria. Irradiation at low temperatures in the frozen state leads to increased cross linking, mutation induction, and lethality at irradiation temperatures between 0 and approximately ?50 ° C. At even lower irradiation temperatures (?130 to ?196 ° C) much larger amounts of energy are required to produce changes in DNA, lethality, and mutation induction. At ?196 ° C bacteria are very resistant to the biological effects of photosensitization and no cross linking of DNA is observed. However, a new pattern of DNA damage is apparent. Irradiation temperature thus affects both the nature and quantity of induced photoproduct and the biological consequences of such changes.     

The interaction of lysozyme with caffeine,theophylline and theobromine in solution

The interactions of lysozyme with caffeine (Caf), theophylline (Tph) and theobromine (Tbr) were investigated using UV–Vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that Caf (Tph or Tbr) caused the fluorescence quenching of lysozyme by the formation of Caf (Tph or Tbr)–lysozyme complex. The binding constants (K _A) and thermodynamic parameters (ΔG°, ΔH°, ΔS°) at two different temperatures, the binding locality, and the binding power were obtained. The results showed that the process of binding Caf (Tph or Tbr) to lysozyme was a spontaneous molecular interaction procedure and the hydrophobic and electrostatic interactions play a major role in stabilizing the complex; The distance r between donor (lysozyme) and acceptor (Caf, Tph or Tbr) was obtained according to fluorescence resonance energy transfer. The effect of Caf (Tph or Tbr) on the conformation of lysozyme was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra techniques. The results showed that the binding of Caf (Tph or Tbr) to lysozyme induced some micro-environmental and conformational changes in lysozyme and disturbed the environment of the polypeptide of lysozyme.     

Trapping at low temperature of oriented chloroplasts: Application to the study of antenna pigments and of the trap of photosystem-1

A technique is described for the preparation of oriented samples from spinach chloroplasts whose linear dichroism is then studied by (flash) absorption spectroscopy. The chloroplasts are suspended in a glycerol-containing medium, oriented in a magnetic field, and slowly cooled in the magnet until the medium is rigid enough to avoid disorientation effects. The absorption spectra in polarized light have been measured at ?50° and ?170°C. They allow the orientation of chlorophyll b to be resolved, and the red transition moment is found to be tilted out of the membrane plane. A study of the flash-induced absorption changes linked to Photosystem-1 activity reveals a progressive evolution of the difference spectra and of the linear dichroism with decreasing temperatures. At ?170°C, the difference spectrum of P700 in the red is well resolved. All transition moments are found to be largely parallel to the membrane plane. The potential use of the technique for other experiments by differential absorption spectroscopy and by EPR techniques is discussed.     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans II. Aggregated complexes-evidence for long-range order.

Aggregated complexes of acridine orange with dermatan and chondroitin sulfates have been studied in aqueous solution by absorption and circular dichroism spectroscopy. Aggregation was found to be favored at high-dye and glycosaminoglycan concentrations, and in solutions where anionic sites of the glycosaminoglycan are effectively complexed with dye. The aggregates can be removed from solution by centrifugation at 27,000 × g for 1 hr or by filtration through a membrane containing pores of 0.1 μm diameter. The aggregated complexes exhibit large-magnitude-ellipticity circular dichroism bands. In addition, the circular dichroism spectrum observed for a solution containing aggregated acridine orange/chondroitin 4-sulfate complexes is nearly a mirror image of that obtained for aggregated acridine orange/dermatan sulfate complexes. Cooperative alterations (sharp transitions) in the circular dichroism ellipticities of the aggregates occur at elevated temperatures, and result in spectroscopically distinct aggregates upon cooling. The circular dichroism properties and temperature effects are attributed to a supramolecular ordering of acridine orange/glycosaminoglycan complexes within the aggregates, which can be reorganized to a more stable form at high temperatures. Mixed aggregates, containing two different glycosaminoglycans, can be formed. The circular dichroism properties of the mixed aggregates also indicate the existence of long-range order in the arrangement of the complexes. Mixed aggregates containing dermatan sulfate and either chondroitin 4-sulfate or chondroitin 6-sulfate resemble pure dermatan sulfate aggregates in circular dichroism characteristics.     

Biophysical probing of the binding properties of a Cu(II) complex with G‐quadruplex DNA: an experimental and computational study

Telomerase inhibition through G‐quadruplex stabilization by small molecules is of great interest as a novel anticancer therapeutic strategy. Here, we show that newly synthesized Cu‐complex binds to G‐quadruplex DNA and induces changes in its stability. This biophysical interaction was investigated in vitro using spectroscopic, voltammetric and computational techniques. The binding constant for this complex to G‐quadruplex using spectroscopic and electrochemical methods is in the order of 10⁵. The binding stoichiometry was investigated using spectroscopic techniques and corresponded to a ratio of 1: 1. Fluorescence titration results reveal that Cu‐complex is quenched in the presence of G‐quadruplex DNA. Analysis of the fluorescence emission at different temperatures shows that ΔH° > 0, ΔS° > 0 and ΔG° < 0, and indicates that hydrophobic interactions played a major role in the binding processes. MD simulation results suggested that this ligand could stabilize the G‐quadruplex structure. An optimized docked model of the G‐quadruplex–ligand mixture confirmed the experimental results. Based on the results, we conclude that Cu‐complex as an anticancer candidate can bind and stabilize the G‐quadruplex DNA structure. Copyright © 2015 John Wiley & Sons, Ltd.     

The importance of fluctuating thermal regimes for repairing chill injuries in the tropical beetle Alphitobius diaperinus (Coleoptera: Tenebrionidae) during exposure to low temperature

Abstract. In this study, the impact of acclimation (1 month at 15 °C vs. breeding at 30 °C) and fluctuating thermal regimes (daily transfers from low temperatures to various higher temperatures for 2 h) on the cold tolerance of the tropical beetle, Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae) was examined. Acclimation increased significantly the duration of survival (Lt₅₀) at a constant 5 °C (7.7 ± 0.3 days to 9.7 ± 0.5 days). Survival of acclimated and nonacclimated beetles increased slightly at alternating temperatures of 5 °C/10 °C or 5 °C/15 °C. When daily transfer to 20 °C was applied, survival (Lt₅₀) was improved markedly (nonacclimated: 15.5 ± 0.7 days, acclimated: 19.6 ± 0.6 days). The higher temperatures may allow progressive repair of injuries, and the effects of chilling may be repaired completely at 25 and 30 °C, a phenomenon recorded here for the first time. It is estimated that the theoretical upper threshold of chill injury (Th) of nonacclimated beetles is 15.1 °C whereas it is shifted down to 11.2 °C in acclimated beetles, which might enable this temperature to allow effective repair of injury.     

Exploring the intercalation binding of tiamulin with DNA using multispectroscopy and a molecular modelling approach

The binding of tiamulin with calf thymus DNA was systematically investigated using multispectroscopy and molecular modelling techniques. For DNA, once tiamulin was added, viscosity (η) and melting temperature (T_m) both exhibited an uptrend. The fluorescence performance of the tiamulin–DNA complex did not change with the ionic strength changes. The binding constant (K_a) of tiamulin for single-stranded DNA (ssDNA, 1.48 × 10⁴ M⁻¹) was obviously higher than that for double-stranded DNA (dsDNA, 9.51 × 10³ M⁻¹) at 291 K. The helix structure became looser and the base stack force became stronger for DNA due to the presence of tiamulin as seen from circular dichroic (CD) spectra. The intercalation binding mode of tiamulin with DNA was disclosed. Molecular modelling also revealed tiamulin inserting into the base pairs with the lowest binding free energy of −18.73 kJ mol⁻¹ using van der Waals forces as well as hydrogen bonds.     

Circular dichroism of aggregated deoxyhemoglobin S and the effect of amino acids.

It is well known that deoxyhemoglobin S (deoxy Hb S) aggregates at 37 °C and that it disaggregates at 1–5 °C. In this study solutions of pure Hb S at concentrations of 20–22 g/100 ml exhibit a normal circular dichroic spectrum in the range 250–650 nm at the temperature 1 °C. However, by the proper manipulation of the following parameters: temperatures of 1, 24 and 37 °C as well as the times required to change temperature and periods of maintaining at a certain temperature, five stages with different circular dichroic spectra can be produced. Not only the dichroic spectra of these stages are different but the kinetic behavior and stability of each of these stages are different. The evidence suggests that the mechanism of aggregation is similar to crystallization; that is, it exhibits a period of nucleation followed by growth. The overall kinetics of circular dichroic changes are described. At representative solution conditions the circular dichroic changes have been compared and found to parallel gel formation with pure Hb S. Also, the effect of certain anti-sickling amino acids (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118) on the minimum Hb S concentration at which circular dichroic changes occur has been studied, and arginine chloride and arginine aspartate were found to raise this minimum concentration appreciably.