Changes In Circular Dichroism Spectra Research Articles

Deciphering the binding mechanism of an anti-cancer phytochemical plumbagin with calf thymus DNA using biophysical and in silico techniques.

Plumbagin (PLM), a plant derivative, is well known for a wide range of therapeutic effects in humans including anti-cancer, anti-inflammatory, anti-oxidant, and anti-microbial. Cytotoxic and genotoxic potential of this phytochemical has been studied which demands further insight. DNA being a major target for several drugs was taken to study against PLM to understand its effects on the cellular system. UV-Vis spectroscopy has indicated the binding of PLM to ctDNA and dye displacement assays have confirmed the formation of PLM-ctDNA complex. The insignificant changes in circular dichroism spectra suggested that PLM is not affecting the structural makeup of the ctDNA, hence the binding could be peripheral and not intercalating. Further, the relative viscosity and minimal change in melting temperature upon the complex formation supported this finding and confirmed the groove binding of PLM. Molecular docking analysis and simulation studies also show PLM as a minor groove binder to DNA and provide details on the interaction dynamics of PLM-DNA complex. Docking followed by a 100ns simulation reveals the negative Gibbs free energy change (∆G = -6.6kcalmol-1), and the formation of a stable complex. The PLM- DNA complex with stable dynamics was further supported by different parameters including RMSD, RMSF, SASA, Rg, and the energy profile of interaction. This study provides an insight into the cytotoxic and genotoxic mechanism of PLM which can be a crucial step forward to exploit its therapeutic potential against several diseases including cancer.

Recognition of ATT Triplex and DNA:RNA Hybrid Structures by Benzothiazole Ligands.

Interactions of an array of nucleic acid structures with a small series of benzothiazole ligands (bis-benzothiazolyl-pyridines—group 1, 2-thienyl/2-benzothienyl-substituted 6-(2-imidazolinyl)benzothiazoles—group 2, and three 2-aryl/heteroaryl-substituted 6-(2-imidazolinyl)benzothiazoles—group 3) were screened by competition dialysis. Due to the involvement of DNA:RNA hybrids and triplex helices in many essential functions in cells, this study’s main aim is to detect benzothiazole-based moieties with selective binding or spectroscopic response to these nucleic structures compared to regular (non-hybrid) DNA and RNA duplexes and single-stranded forms. Complexes of nucleic acids and benzothiazoles, selected by this method, were characterized by UV/Vis, fluorescence and circular dichroism (CD) spectroscopy, isothermal titration calorimetry, and molecular modeling. Two compounds (1 and 6) from groups 1 and 2 demonstrated the highest affinities against 13 nucleic acid structures, while another compound (5) from group 2, despite lower affinities, yielded higher selectivity among studied compounds. Compound 1 significantly inhibited RNase H. Compound 6 could differentiate between B- (binding of 6 dimers inside minor groove) and A-type (intercalation) helices by an induced CD signal, while both 5 and 6 selectively stabilized ATT triplex in regard to AT duplex. Compound 3 induced strong condensation-like changes in CD spectra of AT-rich DNA sequences.

Testing the Potential of the Ferrocene Chromophore as a Circular Dichroism Probe for the Assignment of the Screw-Sense Preference of Tripeptides

A combined strategy utilizing experimental (infrared, nuclear magnetic resonance, circular dichroism, X-ray) and computational (time-dependent density functional theory) techniques is illustrated on tripeptides to investigate their chiroptical properties. Eight stereoisomers of monosubstituted ferrocene, Boc-Pro-Pro-Ala-NHFc, have been prepared and conformationally characterized in depth to reach some guidelines for a future design of ferrocene-based chromophores. The origin of the sign change in circular dichroism spectra near the absorption maximum of a ferrocene chromophore (about 470 nm) is strongly correlated with the sign of the dihedral angle describing deviation of the directly attached amide plane from planarity of the cyclopentadienyl ring. These findings may provide some guidelines for the design of new chiroptical ferrocene-based probes for the assignment of the screw-sense preference of short peptides.

Studies on binding of single-stranded DNA with reduced graphene oxide-silver nanocomposites.

The binding reaction of reduced graphene oxide-silver nanocomposites (rGO-AgNCs) with calf thymus single-stranded DNA (ssDNA) was studied by ultraviolet-visible absorption, fluorescence spectroscopy and circular dichroism (CD), using berberine hemisulphate (BR) dye as a fluorescence probe. The absorbance of ssDNA increases, but the fluorescence intensity is quenched with the addition of rGO-AgNCs. The binding of rGO-AgNCs with ssDNA was able to increase the quenching effects of BR and ssDNA, and induce the changes in CD spectra. All of the evidence indicated that there was a relatively strong interaction between ssDNA and rGO-AgNCs. The data obtained from fluorescence experiments revealed that the quenching process of ssDNA caused by rGO-AgNCs is primarily due to complex formation, i.e. static quenching. The increasing trend of the binding equilibrium constant (Ka) with rising temperature indicated that the binding process was an endothermic reaction. The calculated thermodynamic parameters showed that the binding process was thermodynamically spontaneous, and hydrophobic association played predominant roles in the binding of ssDNA to the surface of rGO-AgNCs.

DNA Cleavage and Trypanosomes Death by a Combination of Alamar Blue and Au(III)

Alamar Blue test indicates toxicity of Au(III), but Au(III) ion itself is not toxic in vitro and does not cleave DNA. However, combination of Alamar Blue and Au(III) cleaved DNA through formation of O2-• and H2O2 and showed high toxicity on Trypanosoma brucei brucei that causes African Sleeping Sickness (Trypanosomiasis) in vitro and in vivo tests. The DNA cleavage study was performed by gel electrophoresis and circular dichroism (CD) techniques. In vitro study was carried out with tissue cultured adapted T. b. brucei 427 strain blood-stream form parasites which were grown in HMI-9 medium with and without Au(III) ion and incubated at 37 and#176;C in 5% CO2. Toxicity of the mixture of Alamar Blue and Au(III) ion was also tested in vivo with mice which were infected intraperitoneally with 4.0 x 104 trypanosomes. Mechanism of DNA cleavage and of toxicity in vitro test is discussed for the mixture of Alamar Blue and Au(III) ion. Alamar Blue has long been used for cell proliferation. Recently it has been found that Alamar Blue induces cleavage of DNA in the presence of Au(III) through the formation of O2-• and H2O2. On the other hand, Alamar Blue alone or Au(III) ion is incapable of DNA cleavage. It has also been observed that in the presence of certain metal ions such as Pd(II), Zn(II), Cd(II) and Pb(II), Alamar Blue is incapable of DNA cleavage. The cleavage study was carried by gel electrophoresis and circular dichroism (CD) techniques. It was observed that Form I and Form II of the pBluescript plasmid DNA were converted into Form III and DNA fragment by gel electrophoresis. The intensity of Form II was gradually decreased with increasing concentration of Au(III) with the completed disappearance at its higher concentration. The inhibitory effect by the superoxide dismutase (SOD) and catalase confirmed the formation of O2-• and H2O2 respectively in the presence of Alamar Blue and Au(III) ion. The drastic change in CD spectra from positive ellipticity to negative ellipticity at 270 nm is also suggested the cleavage of DNA with increasing concentration of Au(III) in the presence of Alamar Blue. The toxic effect of the mixture of Alamar Blue and Au(III) on Trypanosoma brucei brucei [that causes African Sleeping Sickness (Trypanosomiasis)] was investigated and found the IC50 value for aqueous Au(III) was 3.8 x 10-7 M while that for Pd(II) was 1.3 and#215; 10-5 M. However, Alamar Blue alone or Au(III) ion did not show any significant toxic effect on the parasite. The toxic effect of the mixture of Alamar Blue and Au(III) ion was also tested in vivo with mice which were infected intraperitoneally with 4.0 x 104 trypanosomes. Mechanism of DNA cleavage and of toxicity in vitro test is discussed for the mixture of Alamar Blue and Au(III) ion.

DNA Cleavage and Trypanosomes Death by a Combination of Alamar Blue and Au(III)

Alamar Blue test indicates toxicity of Au(III), but Au(III) ion itself is not toxic in vitro and does not cleave DNA. However, combination of Alamar Blue and Au(III) cleaved DNA through formation of O2-• and H2O2 and showed high toxicity on Trypanosoma brucei brucei that causes African Sleeping Sickness (Trypanosomiasis) in vitro and in vivo tests. The DNA cleavage study was performed by gel electrophoresis and circular dichroism (CD) techniques. In vitro study was carried out with tissue cultured adapted T. b. brucei 427 strain blood-stream form parasites which were grown in HMI-9 medium with and without Au(III) ion and incubated at 37 and#176;C in 5% CO2. Toxicity of the mixture of Alamar Blue and Au(III) ion was also tested in vivo with mice which were infected intraperitoneally with 4.0 x 104 trypanosomes. Mechanism of DNA cleavage and of toxicity in vitro test is discussed for the mixture of Alamar Blue and Au(III) ion. Alamar Blue has long been used for cell proliferation. Recently it has been found that Alamar Blue induces cleavage of DNA in the presence of Au(III) through the formation of O2-• and H2O2. On the other hand, Alamar Blue alone or Au(III) ion is incapable of DNA cleavage. It has also been observed that in the presence of certain metal ions such as Pd(II), Zn(II), Cd(II) and Pb(II), Alamar Blue is incapable of DNA cleavage. The cleavage study was carried by gel electrophoresis and circular dichroism (CD) techniques. It was observed that Form I and Form II of the pBluescript plasmid DNA were converted into Form III and DNA fragment by gel electrophoresis. The intensity of Form II was gradually decreased with increasing concentration of Au(III) with the completed disappearance at its higher concentration. The inhibitory effect by the superoxide dismutase (SOD) and catalase confirmed the formation of O2-• and H2O2 respectively in the presence of Alamar Blue and Au(III) ion. The drastic change in CD spectra from positive ellipticity to negative ellipticity at 270 nm is also suggested the cleavage of DNA with increasing concentration of Au(III) in the presence of Alamar Blue. The toxic effect of the mixture of Alamar Blue and Au(III) on Trypanosoma brucei brucei [that causes African Sleeping Sickness (Trypanosomiasis)] was investigated and found the IC50 value for aqueous Au(III) was 3.8 x 10-7 M while that for Pd(II) was 1.3 and#215; 10-5 M. However, Alamar Blue alone or Au(III) ion did not show any significant toxic effect on the parasite. The toxic effect of the mixture of Alamar Blue and Au(III) ion was also tested in vivo with mice which were infected intraperitoneally with 4.0 x 104 trypanosomes. Mechanism of DNA cleavage and of toxicity in vitro test is discussed for the mixture of Alamar Blue and Au(III) ion.

Interaction between Silver Nanoparticles and Two Dehydrogenases: Role of Thiol Groups.

Widely used silver nanoparticles (AgNPs) are readily accessible to biological fluids and then surrounded by proteins. However, interactions between AgNPs and proteins are poorly understood. Two dehydrogenases, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase (MDH), are chosen to investigate these interactions. Ag bound to thiol groups of these enzymes significantly decreases the number of free thiols available. Dose-dependent inhibition of enzyme activities is observed in both AgNPs and Ag+ treatments. Based on the concentration required to inhibit 50% activity, GAPDH and MDH are 24-30 fold more sensitive to Ag+ than to AgNPs suggesting that the measured 4.2% Ag+ containing AgNPs can be responsible for the enzymes inhibition. GAPDH, with a thiol group in its active site, is more sensitive to Ag than MDH, displaying many thiol groups but none in its active site, suggesting that thiol groups at the active site strongly determines the sensitivity of enzymes toward AgNPs. In contrast, the dramatic changes of circular dichroism spectra show that the global secondary structure of MDH under AgNPs treatment is more altered than that of GAPDH. In summary, this study shows that the thiol groups and their location on these dehydrogenases are crucial for the AgNPs effects.

Unusual stability exhibited by (AT)XN12(AT)Y motif associated with high fetal hemoglobin levels

Quasi-palindromic sequences (AT)XN12(AT)Y present in HS2 (hypersensitive site 2) of the human β-globin locus are known to be significantly associated with increased fetal hemoglobin (HbF) levels. High HbF levels in some adults arise due to pathological conditions such as sickle cell disease and β-thalassemia. However, elevated levels of HbF are also associated with a reducing morbidity and mortality in patients with β-thalassemia and thus ameliorate the severity of the disease. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, and circular dichroism (CD) techniques, we demonstrated that it exhibits a hairpin-duplex equilibrium. Intramolecular species (hairpin) were observed in both low and high salt concentrations in gel assay studies displaying the unusual stability of intramolecular species even at the high counter-ion concentration. The unusual stability of hairpin secondary structures was also demonstrated by the monophasic nature of the melting profiles for the oligonucleotides which persisted at low as well as high salt and oligomer concentrations. Change in CD spectra as a function of oligomer concentration indicates that the bimolecular duplex formation is selectively favored over monomolecular hairpin formation at and above 9 µM oligomer concentration. Thus, we hypothesize that imperfect inverted repeat sequence (AT)XN12(AT)Y of HS2 of β-globin gene LCR forms the unusually stable hairpins which may result in the formation of a cruciform structure that may be recruited for binding by various nuclear proteins that could result in elevated HbF levels.Communicated by Ramaswamy H. Sarma.

A two-step heat treatment of cell disruption supernatant enables efficient removal of host cell proteins before chromatographic purification of HBc particles

The thermal stability of HBc particles was systematically investigated for efficient removal of host cell proteins (HCP) by heat treatment before chromatographic step. The HBc particles were found stable up to 80°C for 30 min without any noticeable change in circular dichroism spectra, fluorescence spectra and transmission electron microscope observation. When heating was applied to precipitate the HCP in the cell disruption supernatant of HBc fermentation, the HCP removal effect was more obvious as the temperature went higher. However, a phenomenon was found beyond 70°C where the recovered HBc particles had larger than normal size and molecular weight as observed by dynamic light scattering and multi-angle laser light scattering. Analysis found that the HBc particles possess nanopores which expand with temperature. When the temperature was above 70℃, the pores were large enough for some HCP to penetrate in, but not being able to get out after cooling down. To fully utilize the thermal stability and avoid the interference of HCP entering, a two-step heat treatment strategy was designed. The supernatant was firstly heated up to 60°C for 30 min to precipitate most HCP, then another 30 min at 70°C was used to remove the rest impurities. The two-step heat treatment effectively avoided the HCP entering problem, achieving 85.8% particle recovery and 74.7% purity. With further one-step hydrophobic interaction chromatography, the purity was increased to 99.0% with overall process recovery of 77.7%, considerably higher than those reported in the literature. The same process design was applied to purify three HBc-related products, including OVA-HBc, M2e-HBc and NP-HBc. All recoveries were higher than 50% with purity greater than 97%.

Theoretical Investigation of Single-Molecule Sensing Using Nanotube-Enhanced Circular Dichroism.

First-principles calculations have been used to investigate the potential use of circular dichroism (CD) spectroscopy in single-molecule sensing. Using a real-space implementation of time-dependent density functional theory (TDDFT), several systems involving single-walled carbon nanotubes (SWCNT) and small molecules have been studied to evaluate their CD response. Large induced CD (ICD) effects, differing for each test molecule, were observed in all SWCNT-molecule complexes. As the SWCNT used in this study shows no intrinsic CD response, the ICD spectra are the result of interaction with the small molecules. This finding is general and independent of the (a)chiral nature of the adsorbed molecule. Our results indicate that it is possible to design a system that uses SWCNT for detection of molecules using the change in CD spectrum of the system induced by adsorption of the molecule onto the SWCNT surface.

A Structural and Functional Role for Disulfide Bonds in a Class II Hydrophobin.

Hydrophobins are multifunctional, highly surface active proteins produced in filamentous fungi and can be identified by eight conserved cysteine residues, which form four disulfide bridges. These proteins can be subdivided into two classes based on their hydropathy profiles, solubility, and structures formed upon interfacial assembly. Here, we probe the structural and functional roles of disulfide bonds for a class II hydrophobin in different interfacial contexts by reducing its disulfides with 1,4-dithiothreitol and blocking the free thiols with iodoacetamide and then examining the protein secondary structure, emulsification capability, hydrophobic surface wetting, and solution self-assembly. Changes in circular dichroism spectra upon reduction and blocking of disulfides are consistent with an increase in the level of random coil secondary structure. Emulsification of octane in water using reduced and unreduced forms of class II hydrophobin showed a substantial loss of emulsification ability without disulfides and stable emulsion formation for hydrophobin with disulfides. Additionally, water contact angle measurements performed on polytetrafluoroethylene treated with solutions of reduced and unreduced hydrophobin showed efficient wetting of the hydrophobic surface for unreduced samples only. Lastly, Förster resonance energy transfer (FRET) was used to assess the role of disulfides in self-assembly in solution, and near complete loss of the FRET signal is consistent with a model in which solution self-assembly does not occur after reduction and blocking of the disulfides. From this, we conclude that, in contrast to class I hydrophobins, the disulfides of this class II hydrophobin are required for protein structural stability, surface activity at both liquid-liquid and solid-liquid interfaces, and solution self-assembly.

Circular dichroism spectroscopy of complexes based on semiconductor quantum dots and chlorin e6 molecules

Circular dichroism (CD) spectra of complexes based on ZnS:Mn/ZnS and CdSe/ZnS quantum dots (QDs) and chlorin e6 (Ce6) molecules in dimethyl sulfoxide (DMSO) and in aqueous solutions at different pH levels were investigated. The changes in CD spectra of Ce6 upon its bonding in complex with semiconductor QDs were analyzed. CD spectroscopy allowed us to obtain the CD spectrum of a luminescent Ce6 dimer and to identify a nonluminescent Ce6 aggregate, which is thought to be a tetramer. The dissymmetry factor of the tetramer is 40 times larger than that of the Ce6 monomer. The analysis of the obtained data showed that in complexes with QDs Ce6 can be either in the monomer form or in the form of a nonluminescent tetramer. The interaction of the relatively unstable luminescent Ce6 dimer with QDs leads to its partial monomerization and the formation of complexes with Ce6 in the monomer form. On the basis of time-dependent density functional theory calculations, we performed a geometry model of Ce6 dimer form with corresponding calculated absorption and CD spectra, which are in a qualitative agreement with the experimental data.

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.

Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions.

We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9M urea or 6M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.

Deciphering the groove binding modes of tau-fluvalinate and flumethrin with calf thymus DNA

Tau-fluvalinate (TFL) and flumethrin (FL), widely used in agriculture and a class of synthetic pyrethroid pesticides with a similar structure, may cause a potential security risk. Herein, the modes of binding in vitro of TFL and FL with calf thymus DNA (ctDNA) were characterized by fluorescence, UV–vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy with the aid of viscosity measurements, melting analyses and molecular docking studies. The fluorescence titration indicated that both TFL and FL bound to ctDNA forming complexes through hydrogen bonding and van der Waals forces. The binding constants of TFL and FL with ctDNA were in the range of 104Lmol−1, and FL exhibited a higher binding propensity than TFL. The iodide quenching effect, single/double-stranded DNA effects, and ctDNA melting and viscosity measurements demonstrated that the binding of both TFL and FL to ctDNA was groove mode. The FT-IR analyses suggested the A–T region of the minor groove of ctDNA as the preferential binding for TFL and FL, which was confirmed by the displacement assays with Hoechst 33258 probe, and the molecular docking visualized the specific binding. The changes in CD spectra indicated that both FL and TFL induced the perturbation on the base stacking and helicity of B-DNA, but the disturbance caused by FL was more obvious. Gel electrophoresis analyses indicated that both TFL and FL did not cause significant DNA cleavage. This study provides novel insights into the binding properties of TFL/FL with ctDNA and its toxic mechanisms.

Study on the interaction between ginsenoside Rh2 and calf thymus DNA by spectroscopic techniques.

The interaction between ginsenoside Rh2 (G-Rh2) and calf thymus DNA (ctDNA) was investigated by spectroscopic methods including UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopy, coupled with DNA melting techniques and viscosity measurements. Stern-Volmer plots at different temperatures proved that the quenching mechanism was a static quenching procedure. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -22.83 KJ · mol(-1) and 15.11 J · mol(-1) · K(-1) by van 't Hoff equation, suggesting that hydrophobic force might play a major role in the binding of G-Rh2 to ctDNA. Moreover, the fluorescence quenching study with potassium iodide as quencher indicated that the KSV (Stern-Volmer quenching constant) value for the bound G-Rh2 with ctDNA was lower than the free G-Rh2. The relative viscosity of ctDNA increased with the addition of G-Rh2 and also the ctDNA melting temperature increased in the presence of G-Rh2. Denatured DNA studies showed that quenching by single-stranded DNA was less than that by double-stranded DNA. The observed changes in CD spectra also demonstrated that the intensities of the positive and negative bands decreased with the addition of G-Rh2. The experimental results suggest that G-Rh2 molecules bind to ctDNA via an intercalative binding mode.

Looking at human cytosolic sialidase NEU2 structural features with an interdisciplinary approach.

Circular dichroism (CD) spectra at variable temperatures have been recorded for human cytosolic sialidase NEU2 in buffered water solutions and in the presence of divalent cations. The results show the prevalence of β-strands together with a considerable amount of α-helical structure, while in the solid state, from both previous X-ray diffraction analysis and our CD data on film samples, the content of β-strands is higher. In solution, a significant change in CD spectra occurs with an increase in temperature, related to a decrease in α-helix content and a slight increase in β-strand content. In the same range of temperatures, the enzymatic activity decreases. Although the overall structure of the protein appears to be particularly stable, molecular dynamics simulations performed at various temperatures evidence local conformational changes possibly relevant for explaining the relative lability of enzymatic activity.

Chiral electronic transitions in fluorescent silver clusters stabilized by DNA.

Fluorescent, DNA-stabilized silver clusters are receiving much attention for sequence-selected colors and high quantum yields. However, limited knowledge of cluster structure is constraining further development of these "AgN-DNA" nanomaterials. We report the structurally sensitive, chiroptical activity of four pure AgN-DNA with wide ranging colors. Ubiquitous features in circular dichroism (CD) spectra include a positive dichroic peak overlying the lowest energy absorbance peak and highly anisotropic, negative dichroic peaks at energies well below DNA transitions. Quantum chemical calculations for bare chains of silver atoms with nonplanar curvature also exhibit these striking features, indicating electron flow along a chiral, filamentary metallic path as the origin for low-energy AgN-DNA transitions. Relative to the bare DNA, marked UV changes in CD spectra of AgN-DNA and silver cation-DNA solutions indicate that ionic silver content constrains nucleobase conformation. Changes in solvent composition alone can reorganize cluster structure, reconfiguring chiroptical properties and fluorescence.

Binding properties of butylated hydroxytoluene with calf thymus DNA in vitro

The binding properties of butylated hydroxytoluene (BHT) with calf thymus DNA (ctDNA) in simulated physiological buffer (pH 7.4) were investigated using ethidium bromide (EB) dye as a fluorescence probe by various spectroscopic techniques including UV–vis absorption, fluorescence, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy along with ctDNA melting studies and viscosity measurements. It was found that the binding of BHT to ctDNA could decrease the absorption intensity of ctDNA, significantly increase melting temperature and relative viscosity of ctDNA, and induce the changes in CD spectra. Moreover, the competitive binding studies showed that BHT was able to displace EB from the bound ctDNA–EB complex. All the experimental results indicated that the binding mode between BHT and ctDNA was an intercalation. The association constants between BHT and ctDNA were evaluated to be (4.78±0.04)×103, (2.86±0.02)×103 and (1.80±0.04)×103 L mol−1 at 298, 304, 310K, respectively. Further, the FT-IR analysis revealed that BHT was more prone to interact with adenine and thymine base pairs, and no significant conformational transition of ctDNA occurred. Thermodynamic analysis of the binding data showed that the binding process was primarily driven by hydrogen bonds and van der Waals forces, as the values of the enthalpy change and the entropy change were calculated to be −62.47±0.07kJ mol−1 and −139.22±0.22J mol−1 K−1, respectively.

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.