Methylene Blue Binding Research Articles

Carbonization temperature effects on adsorption performance of metal-organic framework derived nanoporous carbon for removal of methylene blue from wastewater; experimental and spectrometry study

In this study, heat treatment effects on adsorption capabilities of nanoporous carbon particles derived from the metal-organic framework (MOF) were investigated at different temperatures. The carbon nanoparticles were synthesized by one-step carbonization of the metal-organic framework crystals. The results showed that the MOF derived carbons at 1000 °C had an outstanding surface area and micropore volume (1337.9 m2·g−1, 0.72 cm3·g−1) compared to nanoparticles carbonized at 900 °C (1073.9 m2·g−1, 0.58 cm3·g−1) and 800 °C (480.32 m2·g−1, 0.25cm3·g−1). The acid treatment was applied on the 800 °C carbonized specimen due to its lower specific surface area than others, and an increase in its surface area and micropore volume (856.71 m2·g−1, 0.28 cm3·g−1) was observed. The heat treatment at 1000 °C had a significant impact on the adsorption capacity of synthesized MOF derived porous carbon for the removing of methylene blue (MB) (about 2724 mg·g−1) from wastewater. The physicochemical properties of the MOF derived carbon samples were characterized by X-ray diffraction (XRD), HR-TEM, N2 adsorption-desorption, FTIR, Raman Spectroscopy, TGA, and X-ray photoelectron spectroscopy (XPS) analyses. From the XPS results, the chemical environment of oxygen-containing functional groups (C−O and CO) changed by enhancing the temperature that provides sufficient reactive sites for the MB binding during the sorption process. Also, by investigation of the adsorption isotherms of Langmuir, Freundlich Dubinin-Radushkevich (D-R), and Temkin concluded that the D-R isotherm model fitted better with the experimental data than the others. The best kinetic model for the MB adsorption onto the synthesized MOF derived carbons was the pseudo-second-order model. Based on the thermodynamic calculations, the adsorption process is found to be spontaneous and endothermic.

Molecular Interactions and Binding Free Energy of Polydopamine and Methylene Blue: A DFT Study

Polydopamine (PDA) is a kind of mussel-inspired material. It has been applied as a coating and an adsorbent material. As an adsorbent, the PDA microspheres were used to remove methylene blue (MB) in aqueous solution. It was reported that the efficiency of the PDA adsorbent depends on the pH of the solution. Furthermore, in a pH range of 3 - 10, the hydroxyl and amine functional groups of PDA can be either protonated or deprotonated. The change of the net charge of PDA can affect the intermolecular interactions between PDA and MB. It was proposed that both of the electrostatic and p-p interactions should be dominant in the acidic and basic solutions. Therefore, the structural properties and intermolecular interactions of the PDA-MB complex should be investigated. Such an investigation can be useful for the improvement of the PDA microspheres for the other dyes. To get insight into the roles of PDA structure and its role as an interesting adsorbent for MB, the PDA-MB complex formation was carried out at pH 7. The PDA dimers which have six possible structures were selected. The optimization of all PDA dimers and MB was performed in the gas phase at the B3LYP/6-311++G(d,p) calculations. After that, the complex formation of the optimized PDA dimers and MB was performed using the AutoDock Vina V1.1.2. The binding free energy of the PDA dimers and MB was in a range of -3.2 to -3.7 kcal/mol, which indicated that the binding of PDA dimer and MB is spontaneous. The results showed that the p-p interaction between PDA dimer and MB plays a crucial role in the complex formation. Likewise, the sandwich-like structures of the complexes are more stable than the twisted structures.

NMR studies on oligonucleotide – Methylene blue conjugates targeting double-helical nucleic acids

Methylene blue (MB) – nucleic acid interactions are of considerable interest due to the photosensitizing activity of the dye with potential applications in medicine and biotechnology. Covalent attachment of the MB to an oligonucleotide through a flexible heptamethylene linker enabled a positioning of the dye moiety to specific sites through triplex formation with a target duplex. NMR studies demonstrated interactions of MB with the nucleic acids. In sequences with the MB moiety facing the triplex-duplex junction with an alternating CG duplex overhang next to a T·A·T triple-helical tract, proton resonances experienced severe linebroadening upon MB binding and point to kinetically labile complexes with exchange among different binding modes. For sequences with the MB moiety facing a terminal T·A·T base triad of the triplex tract, structural heterogeneity decreased when compared to a triplex without MB attached to the third strand. Also, the thermal stability of the latter construct increased significantly in the presence of MB, indicating external end stacking as predominant binding mode. Without any obvious disruptions of sequential imino-imino NOE contacts within the triplex and duplex tracts, a most favorable intercalation between T·A·T base triples or CG base pairs is not supported by the present data under our experimental conditions.

Acaciain peptidase: The first South American pollen peptidase potentially involved in respiratory allergy.

Acacia caven (Mol.) Molina pollen causes pollinosis in South America. The aim of this work was to isolate, purify, and characterize the proteolytic enzymes of A. caven pollen, and study their influence on allergy. A series of chromatographic steps were applied to purify the proteolytic extract of A. caven pollen. The purified fraction was partially characterized, and then it was assayed on airway bioactive peptides (substance P, vasoactive intestinal peptide, and bradykinin), and peptide degradation was visualized by direct protein sequencing. The cellular detachment of an airway-derived epithelial cell line (A-549) was measured by methylene blue binding assay. The degradation of proteins from intercellular junctions (occludin, claudin, and E-cadherin) was visualized by Western blot. A 75-kDa peptidase, named acaciain peptidase, was purified and classified as a serine peptidase. Acaciain peptidase degraded bioactive peptides involved in the maintenance and recovery of the bronchomotor tone; it caused cellular detachment of A-549 cell line, and degradation of intercellular junction proteins. Acaciain peptidase can alter the integrity of the epithelium barrier, causing cell permeability, increasing the allergic sensitization and exacerbating the overall bronchoconstrictive effect detected in asthmatic lungs. This novel serine peptidase constitutes a relevant therapeutic target in the treatment of allergic disorders.

Carbon dots-sensitized amorphous MoSx photoanode: Sequential electrodeposition preparation and dual amplified photoelectrochemical aptasensing of adenosine

The design and fabrication of high visible-light activated photoelectrode are essential to precisely detect biomolecule in biological system. Herein, an ultrasensitive photoelectrochemical (PEC) aptasensor for specific recognition of adenosine is established based on carbon dots sensitized-amorphous molybdenum sulfide (a-MoSx/CDs) photoanode and dual amplification strategy. The heterostructured photoanode achieved by sequential electrodeposition reveals significantly boosted photocurrent with good stability and repeatability under visible light illumination, giving the credit to highly activated visible light absorption, uniform coverage and good electric contact to the underlying substrate, as well as the energy-band alignment between the two components. By stepwisely immobilizing complementary DNA probe (NH2-DNA) and adenosine aptamer (Apt), followed by methylene blue (MB) binding with the guanine base on Apt, a dual amplified self-powered PEC aptasensor for adenosine detection is constructed. Based on the co-sensitization effect of CDs and MB, ultrasensitive and high-affinitive determination of adenosine is realized over the concentration range of 0.01 nM–1000 nM at 0 V (vs. SCE), with satisfactory stability and reproducibility. The detection limit is as low as 3.3 pM, demonstrating a performance even surpassing most of the sensors reported so far. The prospective application of the co-sensitized a-MoSx photoanode for ultrasensitive aptasensing is highlighted in this work.

A new sensing strategy based on thymine bases–Hg2+–methylene blue coordination on the electrospun PES–QDs platform for detection of Hg2+ in fruit juice samples

A novel label-free and highly sensitive electrochemical aptasensor for highly sensitive mercury ion detection has been successfully fabricated in fruit juice samples using of electrospun nanofibers polyethersulfone and quantum dots (NFs–QDs) at the surface of carbon paste electrode (CPE). In addition to the T bases used in the designed aptasensor which can be strong and specific binding to the Hg2+, the methylene blue (MB) also can interact specifically with the Hg2+ (T–Hg2+–MB), as well as interact with the guanine bases in the DNA aptamer as an receiver site for interaction. According to this, the proposed sensor has more receiver sites for interaction with the Hg2+. Here, the electrochemical signal of MB was employed for Hg2+ measurements which this signal was amplified by NFs–QDs nanocomposites. The Apt–MB binding event easily investigated by the peak current variations in Apt–MB1–Hg2+–MB2/NFs–QDs/CPE through cyclic voltammetry, differential pulse voltammetry (DPV) measurement. Also, this study was evaluated with electrochemical impedance spectroscopy. Under optimized conditions, the constructed aptasensor illustrated a wide linear range using the DPVs, from 0.1 to 150 nM and an excellent low detection limit of 0.02 nM. The quality of carefully choosing, an excellent stability and specificity sensitivity of the designed aptasensor were accredited by spiked juice samples as real sample. Moreover, the aptasensor exhibits the good reproducibility as well as has high selectivity to other cations. The recoveries of the Hg2+ assay of the fruit juice samples were obtained satisfactorily which can use for measurement of Hg2+ in the laboratory.

Effective adsorptive performance of Fe3O4@SiO2 core shell spheres for methylene blue: kinetics, isotherm and mechanism

In this study, Fe3O4 nanoparticles (NPs) as a core surrounded with thick and tunable SiO2 shells were successfully synthesized and characterized by means of N2-isotherm, scanning electron microscopy (SEM), X-ray diffraction (XRD), transmission electron microscopy (TEM), fourier transform infrared (FT-IR) and UV–Vis diffuse reflection spectroscopy (UV–Vis DRS) techniques. The characterization results revealed that well-structured SiO2 shell of 105 nm thick is uniformly formed around Fe3O4 (NPs size of 500 nm) surface. The application of the synthesized core–shell structures was investigated for methylene blue (MB), rhodamine B (Rh B) and methyl orange (MO) toxic dyes adsorptive removal from water by batch adsorption methods and different adsorption parameters such as time, pH, material dosage, concentration of solution and different dyes were optimized. The highest adsorption capacity (123 mg g−1) of MB dye was achieved on Fe3O4@SiO2. The isotherms and kinetic models exhibited that MB adsorption values are well described by Freundlich isotherm and pseudo-first-order kinetics models. The adsorptive binding of MB with Fe3O4@SiO2 was directed through electrostatic interaction and size filter effect. After regeneration, approximately 100% MB dye adsorption capacity was recovered. Thus, we can say that the Fe3O4@SiO2 is an outstanding material for dyes removal from water.

Nigella sativa seed based nanocomposite-MnO2/BC: An antibacterial material for photocatalytic degradation, and adsorptive removal of Methylene blue from water

Antimicrobial Nigella sativa seed-based nanocomposite, MnO2/BC, was synthesized and utilized for the water purification through adsorption, and the photocatalytic degradation. MnO2/BC was prepared by co-precipitation method, and characterized using FT-IR, XRD, SEM, TEM, TGA, and DSC techniques. The composite was investigated for inhibition of bacterial cells growth. FT-IR spectrum indicated the presence of oxygenous groups on the surface; TGA and DSC showed thermal degradation; and XRD, SEM, and TEM investigations indicated amorphous, and porous nature of MnO2/BC having particle size of 190–220 nm. The nanocomposite inhibited the growth of both Gram-positive and Gram-negative bacteria cells in water. The adsorption of Methylene blue from water was investigated in batch method in terms of amount of MnO2/BC, dye concentration, pH, time, and temperature. 1.0 g L−1 of MnO2/BC removed more than 98% of Methylene blue from aqueous solution having concentration of 10 mg L−1 and pH 7.0 at 27 °C. The maximum Langmuir adsorption capacity of MnO2/BC was 185.185 mg g−1 at 45 °C. The adsorption was an endothermic process which obeyed Freundlich isotherm, and pseudo-second order kinetics. Therefore, the Methylene blue binding onto MnO2/BC surface was site-specific partially through the weak hydrogen bonding, and electrostatic interactions. The photocatalytic activity of MnO2/BC has been investigated by degrading the Methylene blue molecules/ions in water under the sunlight and 85% of degradation was achieved during 120 min irradiation. The dye was desorbed at lower pH and regenerated MnO2/BC was used for second cycle of Methylene blue adsorption. The results obtained for this study are much better than the previous Methylene blue adsorption studies with acid washed Black cumin seeds and MnFe2O4/BC for which the capacities were 73.529 mg g−1 and 10.070 mg g−1 at 27 °C, respectively (J. Mol. liq. 2018a, 264, 275–284; J. Clean. Prod. 2018a, 200, 996–1008).

Competitive sorption of lead and methylene blue onto black soil and their interaction with dissolved organic matter using two-dimensional correlation analyses

This study investigated the competitive sorption of black soil to adsorb Pb(II) and methylene blue (MB) from multi-contaminated soils. According to the experimental data, the process of adsorption can be clearly explained by pseudo-second-order kinetic equation. Both single and binary systems of the adsorption isotherms had a good fit with Langmuir models. The maximal adsorption abilities of Pb(II) and MB acquired from binary systems sorption were attenuated compared to those from the single system (Pb(II): 77.70 > 65.96 mg g−1; MB: 242.31 > 222.36 mg g−1). Pb(II) and MB can inhibit each other's sorption ability. A combination of three-dimensional excitation-emission matrix (3D-EEM), synchronous fluorescence spectra as well as two-dimensional correlation spectroscopy (2D-COS) were employed to determine the binding of dissolved organic matter (DOM) for Pb(II) and MB during soil sorption process. As a result, 3D-EEM implicated that the two main composes of DOM were humic acid-like substances and the fluorescence of DOM specimens were gradually diminished with increasing concentrations of Pb(II) and MB. According to synchronous fluorescence spectra, static quenching of Pb(II) and MB mainly led to fluorescence quenching. Specifically, fluorescence-2D-COS implicated that Pb(II) and MB bound to fluorescence in the following sequence: the earlier occurrence of the humic-like fraction compared to that of protein-like fraction. FTIR-2D-COS results concluded that the structural change sequence of DOM by Pb(II) binding followed the order: 1700＞863＞1332＞1529＞1200＞1086 cm−1 and the sequence of the MB binding affinities followed the order: 1520＞1399＞1345＞1152＞1602＞993＞881 cm−1. These findings would be beneficial to understand the mechanism of adsorb multi-component systems and have the potential to contribute significance to the interaction mechanism of multi-component with soil DOM at the molecular level.

Binding Kinetics of Methylene Blue on Monolayer Graphene Investigated by Multiparameter Surface Plasmon Resonance.

In this paper, we study the interaction of a small dye molecule, namely, methylene blue (MB) with graphene surfaces using surface plasmon resonance (SPR). We show that by utilizing all of the parameters of the SPR angular dip and exploiting the fact that MB absorbs light at the operating wavelength, it is possible to detect the binding of small molecules that would otherwise not give a significant signal. The binding of MB to unmodified graphene is found to be stronger than that for gold. By studying the interaction at modified surfaces, we demonstrate that electrostatic effects play a dominant role in the binding of MB on to graphene. Furthermore, following the binding kinetics at various concentrations allows us to estimate apparent equilibrium binding and rate constants for the interaction of MB with graphene.

Picomolar sensitive and SNP-selective “Off-On” hairpin genosensor based on structure-tunable redox indicator signals

A robust and sensitive electrochemical assay for chrononocoulometric detection of nucleic acids at a single nucleotide polymorphism (SNP) level has been developed. The assay exploits hybridization-induced conformational switching of gold-tethered TP53-specific 33-mer and truncated 20-mer hairpin DNA probes and methylene blue (MB) as an intercalating redox indicator. We show that by fine tuning of MB-DNA intercations the enhanced binding of MB to hybrids formed with a cancer-biomarker sequence can be achieved, and that results in robust “off-on” sensing of hybridization, while the stem-loop probe design allows minimized, independent of the DNA length background signals. Both DNA probes were sensitive to the presence of SNP in the targeted DNA sequence already at 10 pM. DNA levels, and the robust “off-on” discrimination of 10 pM perfectly-matched DNA from 50 nM SNP-containing DNA was achieved by time-adjusted chronocoulometry. This label-free hairpin DNA strategy allows systematic design of DNA assays for fast, robust and inexpensive genetic analysis in excessive mixtures of structurally-related DNA sequences and was used for specific analysis of prostate-cancer-realted cellular microRNA in total RNA samples isolated from LNCaP and BPH1 cells.

Comparative Study of Toluidine Blue O and Methylene Blue Binding to Lysozyme and Their Inhibitory Effects on Protein Aggregation.

A comparative binding interaction of toluidine blue O (TBO) and methylene blue (MB) with lysozyme was investigated by multifaceted biophysical approaches as well as from the aspects of in silico biophysics. The bindings were static, and it occurred via ground-state complex formation as confirmed from time-resolved fluorescence experiments. From steady-state fluorescence and anisotropy, binding constants were calculated, and it was found that TBO binds more effectively than MB. Synchronous fluorescence spectra revealed that binding of dyes to lysozyme causes polarity changes around the tryptophan (Trp) moiety, most likely at Trp 62 and 63. Calorimetric titration also depicts the higher binding affinity of TBO over MB, and the interactions were exothermic and entropy-driven. In silico studies revealed the potential binding pockets in lysozyme and the participation of residues Trp 62 and 63 in ligand binding. Furthermore, calculations of thermodynamic parameters from the theoretical docking studies were in compliance with experimental observations. Moreover, an inhibitory effect of these dyes to lysozyme fibrillogenesis was examined, and the morphology of the formed fibril was scanned by atomic force microscopy imaging. TBO was observed to exhibit higher potential in inhibiting the fibrillogenesis than MB, and this phenomenon stands out as a promising antiamyloid therapeutic strategy.

A sensitive, label-free electrochemical detection of telomerase activity without modification or immobilization

Telomerase has become one of the most typical tumor marker because it is closely related to cancers. In this paper, a simple label-free electrochemical detection of telomerase activity by using methylene blue (MB) as a G-quadruplex binding probe was proposed, avoiding commonly used complex label procedures, nano-probe synthesis, complicated electrode modification, probe immobilization or signal amplification. In the presence of telomerase substrate (TS) primer, the binding of MB on primer was weak. When repeats of (TTAGGG) were extended on the TS primer under the action of telomerase, they formed multiple G-quadruplexes with the help of K+. As a result, a large amount of MB bounded to multiple G-quadruplexes because they have more strong interaction with G-quadruplexes than TS primer. As a result, the diffusion current of MB decreased sharply, which was strongly dependent on the telomerase activity. The DPV current change has a linear correlation with the logarithm of HeLa cell number in the range of 10–10,000 cells, with the detection limit of 3 cells. The high sensitivity was due to the formed multiple G-quadruplexes. Using indium tin oxide (ITO) as working electrode without modification ensured the good reproducibility of the method. The method was also simple, rapid, and has been successfully applied in the telomerase activity detection in urine with good selectivity and reproducibility, which is significant for cancer diagnosis, anticancer drugs screening, and cancer therapy evaluation.

Joint interaction of ethidium bromide and methylene blue with DNA. The effect of ionic strength on binding thermodynamic parameters

Large amount of data of experimental and theoretical studies have shown that ethidium bromide (EtBr) and methylene blue (MB) may bind to nucleic acids via three modes: intercalation between two adjacent base pairs, insertion into the plane between neighboring bases in the same strand (semi-intercalation), and outside binding with negatively charged backbone phosphate groups. The aim of the given research is to examine the behavior of these two ligands at both separate and joint DNA binding. The obtained experimental data show that the effect of simultaneous binding of EtBr and MB on double-stranded DNA has a non-additive effect of separate binding. The analyses of the melting thermodynamic parameters of DNA complexes with two bound ligands suggest competitive mechanism of interaction.

Magnetite/Polymer Brush Nanocomposites with Switchable Uptake Behavior Toward Methylene Blue.

The grafting from approach was used to prepare pH-responsive polyacid brushes using poly(itaconic acid) (PIA) and poly(acrylic acid) (PAA) at the amine functional groups of chitosan. Hybrid materials consisting of polymer brushes and magnetite nanoparticles (MNPs) were also prepared. The products were structurally characterized and displayed reversible pH-responsive behavior and controlled adsorption/desorption of methylene blue (MB). Switchable binding of MB involves cooperative effects due to conformational changes of brushes and swelling phenomena in solution which arise from response to changes in pH. Above the pKa, magnetic nanocomposites (MNCs) are deprotonated and display enhanced electrostatic interactions with high MB removal efficiency (>99%). Below the pKa, MNCs undergo self-assembly and release the cationic dye. The switchable binding of MB and the structure of the polymer brush between collapsed and extended forms relate to changes in osmotic pressure due to reversible ionization of acid groups at variable pH. Reversible adsorption-desorption with variable binding affinity and regeneration ability was demonstrated after five cycles.

Binding of methylene blue onto Langmuir monolayers representing cell membranes may explain its efficiency as photosensitizer in photodynamic therapy.

We provide evidence for the electrostatic interactions between the cationic photosensitizer methylene blue (MB) and cell membrane models represented by neat and mixed Langmuir monolayers of dioleylphosphatidylcholine (DOPC) and 1,1',2,2'-tetraoleoylcardiolipin (CL). From surface pressure measurements, MB was found to adsorb strongly and expand CL-containing monolayers, while it caused an apparent decreasing in molecular area on neat DOPC monolayer. The binding site of MB could be inferred from data with the surface-specific polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) technique, where changes induced by MB were observed in the vibrational modes of the phosphate groups of both CL and DOPC. The incorporation of MB also affected the carbonyl groups and the packing of the alkyl chains, thus indicating that MB binding site favors singlet oxygen generation close to the double bonds in the alkyl chains, an important requirement for photodynamic efficiency. Significantly, the data presented here demonstrate that MB may act in membranes composed by PCs, such as mammalian plasma membranes, and in those containing CL, as in bacterial and inner mitochondrial membranes.

Studies of interaction between terbium(III)-deferasirox and double helix DNA by spectral and electrochemical methods

DNA binding studies of terbium(III)-deferasirox (Tb3+-DFX) complex were monitored to understand the reaction mechanism and introduce a new probe for the assay of DNA. In the present work, UV absorption spectrophotometry, fluorescence spectroscopy, circular dichroism (CD), cyclic voltammetry (CV) and viscosity measurement were employed to study the interactions of Tb3+-DFX with calf thymus DNA (ctDNA). The binding of Tb3+-DFX complex to ctDNA showed a hyperchromic effect in the absorption spectra and the increase in fluorescence quenching effect (amount) of Tb3+-DFX complex in the presence of ctDNA. The binding constants (Kb) for the complex with ctDNA were estimated to be 1.8×104M−1 through UV absorption spectrophotometry and fluorescence spectroscopy. Upon addition of the complex, clear decreases were observed in the viscosity of ctDNA. The CD spectra indicated that there are certain detectable conformational changes in the DNA double helix when the complex was added. The CV method showed that both anodic and cathodic peak potentials of Tb3+-DFX complex showed negative shifts on the addition of the ctDNA. Further, competitive methylene blue binding studies with fluorescence spectroscopy have shown that the complex can bind to ctDNA through nonintercalative mode. The experimental results suggest that Tb3+-DFX complex binds to DNA via groove binding and/or electrostatic binding mode.

Fabrication and Characterization of Nanotemplated Carbon Monolithic Material

A novel hierarchical nanotemplated carbon monolithic rod (NTCM) was prepared using a novel facile nanotemplating approach. The NTCM was obtained using C60-fullerene modified silica gels as hard templates, which were embedded in a phenolic resin containing a metal catalyst for localized graphitization, followed by bulk carbonization, and template and catalyst removal. TEM, SEM, and BET measurements revealed that NTCM possessed an integrated open hierarchical porous structure, with a trimodal pore distribution. This porous material also possessed a high mesopore volume and narrow mesopore size distribution. During the course of carbonization, the C60 conjugated to aminated silica was partly decomposed, leading to the formation of micropores. The Raman signature of NTCM was very similar to that of multiwalled carbon nanotubes as exemplified by three major peaks as commonly observed for other carbon materials, i.e., the sp3 and sp2 carbon phases coexisted in the sample. Surface area measurements were obtained using both nitrogen adsorption/desorption isotherms (BET) and with a methylene blue binding assay, with BET results showing the NTCM material possessed an average specific surface area of 435 m2 g(-1), compared to an area of 372 m2 g(-1) obtained using the methylene blue assay. Electrochemical studies using NTCM modified glassy carbon or boron doped diamond (BDD) electrodes displayed quasi-reversible oxidation/reduction with ferricyanide. In addition, the BDD electrode modified with NTCM was able to detect hydrogen peroxide with a detection limit of below 300 nM, whereas the pristine BDD electrode was not responsive to this target compound.

93 Spectroscopic investigation of methylene blue binding to DNA

The interaction of methylene blue (MB) with DNA has been investigated by UV absorption spectra, Fluorescence spectra and UV-melting method. Analysis of the results of the melting experiments shows that melting temperature (T m) of the complexes increases with the [total ligand]: DNA ratio (r) at two concentrations of Na+ (2 mM Na+ and 20 mM Na+) providing support for conclusion that MB is a stabilizer of DNA helix structure. By contrast, the shapes of dependences of width of transition (ΔT) on r at low and high [Na+] are different which points to the existence of different types of binding modes of MB with DNA. UV-spectroscopy experiments and fluorescence spectra indicated that the binding modes of MB with DNA depended on r. At high r (r > 0.25), remarkable hypochromic effect with no shift of λ max in the absorption spectra of MB was observed. The fluorescence of MB was quenched which indicated that MB was bound to phosphate groups of DNA by electrostatic interaction. At low r ratios (r < 0.2), the absorption spectra of MB upon increasing the concentration of DNA showed gradually decrease in the peak intensities with a red shift. This phenomenon is usually associated with molecular intercalation into the base stack of the ds-DNA. Using the Scatchard’s model, the complex formation constants for MB with DNA were determined: the binding constant K ≈ 6.5 × 105 and binding site size n ≈ 4. Obtained data are not typical for intercalation model of ligands to DNA. Moreover, comparison between these data and our early experimental results of interaction of ethidium bromide with DNA made it possible to suggest that this binding type of MB is, more probably, semi-intercalation mode (Vardevanyan et al., 2003). This conclusion is in accordance with the analysis of the model structures of MB–DNA complexes which clearly shows the importance of solvent contributions in suggested structural form (Tong et al., 2010).

Binding of methylene blue to a surface cleft inhibits the oligomerization and fibrillization of prion protein

Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.