safranine-t and phenosafranine

The present study demonstrates a spectroscopic study on the interaction of two phenazinium-based cationic photosensitizers, namely, phenosafranin (PSF) and safranin-O (SO) with human hemoglobin (Hb) with particular emphasis on exploring the effects of pH and chemical structures of the dye molecules on the binding phenomenon. The protein (Hb) undergoes complex conformational transitions depending on the medium pH. The dye molecules exhibit a prominent fluorescence quenching following interaction with Hb under various experimental conditions (pH 3.5, 7.4, and 9.0). Our combined steady-state and time-resolved spectroscopic results provide persuasive evidence for static quenching mechanism showing that the dye:Hb interaction proceeds through ground-state complex formation. The meticulous investigations on the pH-dependence of the interaction of the dye molecules with the protein reveal a relatively strong binding of PSF as well as SO with Hb at physiological pH and alkaline pH, while the binding is weaker at acidic pH at which Hb predominantly exists as monomeric units. The binding constant for PSF:Hb interaction is K(PSF:Hb) = (1.09 ± 0.06) × 10

Topics: Binding Sites; Circular Dichroism; Hemoglobins; Humans; Hydrogen-Ion Concentration; Molecular Docking Simulation; Phenazines; Photosensitizing Agents; Protein Binding; Protein Structure, Tertiary; Spectrometry, Fluorescence

The present work is focused on exploring the interaction of two phenazinium-based biological photosensitizers, phenosafranin (PSF) and safranin-O (SO), with human serum albumin (HSA), with particular emphasis on the physiologically significant NB conformational transition of the protein on the dye:HSA interaction. In addition, the presence of methyl substitution on the planar phenazinium ring in SO paves way for looking into the effect of simple chemical manipulation (that is, methyl substitution on the dye nucleus) on the dye:protein interaction behavior as a function of various (pH-induced) isoforms of HSA. Our results reveal a significantly stronger binding interaction of SO with the B isoform of HSA (at pH9.0) compared to that with the N isoform (at pH7.4). On the contrary, the PSF:HSA interaction is found to be reasonably insensitive to the aforesaid conformational transition of HSA. However, the probable binding location of both the dye molecules (PSF and SO) is found to be within the protein scaffolds (domain IB). This is further quantified from the modulation of fluorescence decay behavior of the dyes within the protein scaffolds. It is important to note that the rotational relaxation behavior of the protein-bound dyes reveals an unusual 'dip-rise-dip', an observation not reported earlier. Such unusual anisotropy decay is meticulously analyzed by an associated (or multicomponent) exponential decay model which emphasizes on the fractional contributions from differential classes of fluorophore populations characterized by the fast (due to unbound or solvent exposed part of the fluorophore) and slow (due to embedded or bound part) motions, in combination with their different local mobilities. Furthermore, the translational diffusion of the dye molecules in the presence of the protein in different isoforms (N-form or B-form) at a single molecule level is also measured by Fluorescence Correlation Spectroscopy (FCS).

Topics: Fluorescent Dyes; Humans; Models, Molecular; Molecular Conformation; Phenazines; Photosensitizing Agents; Protein Binding; Protein Isoforms; Serum Albumin; Structure-Activity Relationship

RNA targeting through small molecules that can selectively bind specific RNA structures is an important current strategy in therapeutic drug development. Towards this strategy a comparative study on the interaction of two phenazinium dyes, safranine-O and phenosafranine to double stranded RNAs, poly(I).poly(C), poly(A).poly(U) and poly(C).poly(G) was performed. Spectrophotometric and spectrofluorimetric studies revealed non-cooperative binding of the dyes to the duplex RNA with binding constants of the order 10(5)M(-1) with a higher affinity of safranine-O to poly(I).poly(C) followed by poly(A).poly(U) and poly(C).poly(G). Anisotropy and fluorescence quenching results confirmed an intercalation mode of binding for the dyes on these RNAs. Binding induced conformational changes in the RNA polynucleotides were revealed from circular dichroism data. Thermal melting study and DSC experiments demonstrated stabilization of dye-RNA complexes. Calorimetric studies revealed that the binding was accompanied by a large positive entropy term with a small negative enthalpy contributions. Significant hydrophobic forces in the complexation of the double stranded RNAs with the dyes were confirmed from the negative heat capacity changes. Enthalpy-entropy compensation was also observed in the binding. Parsing of the Gibbs energy suggested a larger non-electrostatic contribution in all the cases. The results presented here may be helpful to design new types of RNA-based therapeutic agents.

Topics: Calorimetry; Calorimetry, Differential Scanning; Circular Dichroism; Coloring Agents; Entropy; Intercalating Agents; Nucleic Acid Conformation; Nucleic Acid Denaturation; Osmolar Concentration; Phenazines; Polynucleotides; RNA, Double-Stranded; Spectrometry, Fluorescence; Spectrophotometry; Transition Temperature; Ultraviolet Rays

The interaction of phenazinium dyes, safranine O and phenosafranine with single stranded polyadenylic acid was studied using spectroscopic viscometric and calorimetric techniques. Both dyes bind to polyadenylic acid strongly with association constant of the order of 10(5)M(-1). Safranine O showed higher affinity over phenosafranine. The binding induced conformational changes in polyadenylic acid, but the extent of change was much higher with safranine O. The bound safranine O molecules acquired strong induced circular dichroism spectra compared to the weak induced circular dichroism of phenosafranine. Fluorescence polarization, iodide quenching, viscosity results and energy transfer from bases to bound dyes suggested intercalation of the dye molecules to polyadenylic acid structure. The binding was entropy driven in both the cases. Circular dichroism and optical melting studies revealed cooperative melting profiles for dye-polyadenylic acid complexes that provided evidence for the formation of self-structured polyadenylic acid on dye binding. This structural reorganization was further confirmed by differential scanning calorimetry results.

Topics: Calorimetry, Differential Scanning; Circular Dichroism; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Intercalating Agents; Nucleic Acid Conformation; Phenazines; Poly A; Spectrophotometry; Viscosity

Interaction of phenosafranin and safranin O with double stranded, heat denatured and single stranded calf thymus DNA has been studied by fluorescence, absorbance and circular dichroic techniques. Binding to the double stranded and heat denatured DNA conformations induced strong quenching in the fluorescence spectra of both dyes. Linear Scatchard plots indicated the binding to be of one type and the affinity evaluated to be of the order of 10(5) M(-1) with double stranded and heat denatured DNAs. Fluorescence quenching was much weaker with the single stranded DNA and the binding affinity was one order lower. Ferrocyanide quenching studies revealed that the fluorescence emission of the dye molecules bound to the double stranded and heat denatured DNAs was quenched much less compared to that bound to the single stranded DNA. Further, there was significant emission polarization for the bound dyes and strong energy transfer from the DNA base pairs to the dye molecules indicating intercalative binding. Salt dependence of the binding phenomenon revealed that electrostatic forces have significant role in the binding process. The intercalation of these molecules to double stranded and heat denatured DNA and simple stacking to single strands was proved by these fluorescence techniques. Support to the fluorescence results have been derived from absorption and circular dichroic results. Phenosafranin was revealed to be a stronger binding species compared to safranin O.

Topics: Animals; Cattle; DNA; Hot Temperature; Nucleic Acid Denaturation; Phenazines; Spectrometry, Fluorescence

The base specificity and energetics of DNA binding of the phenazinium dyes phenosafranine and safranine-O have been studied using various biophysical tools. The guanine-cytosine base specificity of both compounds was established from binding affinity values and competition dialysis results and also from circular dichroism, thermal melting, and calorimetric studies. Both dyes bind to DNA with affinity of the order of 10(5) M(-1), but the values are significantly higher for the guanine-cytosine rich DNAs over adenine-thymine rich ones and for phenosafranine over safranine-O. Calorimetric studies revealed that the binding reactions were exothermic and favoured by negative enthalpy as well as predominantly large positive entropy contributions. The temperature dependence of enthalpy changes yielded negative heat capacity values, which were higher for phenosafranine, compared to safranine-O, suggesting substantial contribution from hydrophobic forces in the binding process. Enthalpy-entropy compensation behaviour was also observed for the binding of both dyes to DNAs, revealing the molecular aspects of the interaction. Taken together, the spectroscopic and calorimetric data reflect clearly the guanine-cytosine base specificity of these molecules and a stronger DNA binding of PSF over SO. The results also provide some insights into the role of a bulkier substituent in the phenazinium ring in the binding process.

Topics: Animals; Base Pairing; Binding Sites; Calorimetry, Differential Scanning; Cattle; Circular Dichroism; Clostridium perfringens; Coloring Agents; DNA; DNA, Bacterial; Micrococcus; Phenazines; Spectrometry, Fluorescence; Thermodynamics

The sequence selectivity of the DNA binding of the phenazinium dyes phenosafranin and safranin O have been investigated with four sequence-specific deoxyribopolynucleotides from spectroscopic and calorimetric studies. The alternating guanine-cytosine sequence selectivity of the dyes has been revealed from binding affinity values, circular dichroism, thermal melting, competition dialysis, and calorimetric results. The binding affinities of both the dyes to the polynucleotides were of the order of 10(5) M(-1), but the values were higher for the guanine-cytosine polynucleotides over adenine-thymine ones. Phenosafranin had a higher binding affinity compared to safranin O. Isothermal titration calorimetric studies revealed that the binding reactions were exothermic and favored by negative enthalpy and predominantly large positive entropy contributions in all cases except poly(dA)·poly(dT) where the profile was anomalous. Although charged, nonpolyelectrolytic contribution was revealed to be dominant to the free energy of binding. The negative heat capacity values obtained from the temperature dependence of enthalpy changes, which were higher for phenosafranin compared to safranin O, suggested significant hydrophobic contribution to the binding process. In aggregate, the data presents evidence for the alternating guanine-cytosine base pair selectivity of these phenazinium dyes and a stronger binding of phenosafranin over safranin O.

Topics: Base Sequence; Calorimetry, Differential Scanning; Coloring Agents; Cytosine; Guanine; Molecular Structure; Phenazines; Polydeoxyribonucleotides; Spectrum Analysis; Thermodynamics

Absorption, steady-state fluorescence, steady-state fluorescence anisotropy, and intrinsic and induced circular dichroism (CD) have been exploited to explore the binding of calf thymus DNA (ctDNA) with three cationic phenazinium dyes, viz., phenosafranin (PSF), safranin-T (ST), and safranin-O (SO). The absorption and fluorescence spectra of all the three dyes reflect significant modifications upon interaction with the DNA. A comparative study of the dyes with respect to modification of fluorescence and fluorescence anisotropy upon binding, effect of urea, iodide-induced fluorescence quenching, and CD measurements reveal that the dyes bind to the ctDNA principally in an intercalative fashion. The effect of ionic strength indicates that electrostatic attraction between the cationic dyes and ctDNA is also an important component of the dye-DNA interaction. Intrinsic and induced CD studies help to assess the structural effects of dyes binding to DNA and confirm the intercalative mode of binding as suggested by fluorescence and other studies. Finally it is proposed that dyes with bulkier substitutions are intercalated into the DNA to a lesser extent.

Topics: Absorption; Animals; Cattle; Coloring Agents; DNA; Fluorescence; Fluorescence Polarization; Intercalating Agents; Osmolar Concentration; Phenazines; Static Electricity; Urea

Absorption, fluorescence, and fluorescence excitation spectral studies of two planar, cationic phenazinium dyes, namely, phenosafranin (PSF) and safranin-T (ST), have been performed in protic and aprotic polar solvents. The studies reveal the formation of both J- and H-aggregates in concentrated solutions. The planarity of the phenazinium skeleton and the presence of a positive charge are attributed to be the driving force for this aggregation behavior. The aggregates are established to be dimers only. The positive inductive effect of the methyl substituents in safranin-T augments the aggregation process. The experiments reveal that for both dyes, the polar protic solvent favors the aggregation process more than the aprotic solvent.

Topics: Absorption; Acetonitriles; Coloring Agents; Fluorescence; Phenazines; Spectrum Analysis; Water

The absorption spectra of phenazine dyes such as phenosafranin (PSF), safranin-O (Saf-O), and safranin-T (Saf-T) in aqueous solution of Triton X-100 (TX-100) show that phenazine dyes form 1:1 charge-transfer (CT) or electron-donor-acceptor (EDA) complex with TX-100. The photogalvanic and photoconductivity studies also support the above interaction. From the thermodynamic, spectrophotometric and photophysical parameters of these complexes, the abilities of dyes to accept electron are found to be in the order: PSF > Saf-O > Saf-T. There is a good correlation among the spectral and thermodynamic properties of these complexes.

Topics: Coloring Agents; Electrochemistry; Octoxynol; Phenazines; Solutions; Spectrophotometry; Surface-Active Agents; Thermodynamics; Water

The investigation reports on the use of safranine-SO2 and phenosafranine-SO2, prepared with N HCl or oxalic acid plus potassium metabisulphite, for staining rat liver sections following Feulgen procedure. It has been found that optimum staining of DNA-aldehyde molecules is possible with safranine-SO2 and phenosafranine-SO2, prepared with N HCl and potassium metabisulphite, upto a duration of one week after the preparation of the dye-reagents. Thereafter, staining intensity of the nuclei produced by the dye-reagents is gradually diminished. Staining of acid-hydrolysed sections is also possible with aqueous solutions of these dyes. Moreover, DNA-phosphate groups can also be stained with aqueous solutions of these dyes after selective extraction of RNA with cold phosphoric acid. The in situ absorption spectra of nuclei, stained for DNA-aldehyde molecules with safranine-SO2, phenosafranine-SO2 and aqueous solutions of these dyes, have been presented in this paper. Also presented herein are absorption data of nuclei stained with these dyes after selective extraction of RNA. It has been found that absorption-peaks of nuclei stained differently are different from one another. The implications of these findings have been discussed.

Topics: Aldehydes; Animals; Cell Nucleus; DNA; Hydrogen-Ion Concentration; Liver; Phenazines; Phosphates; Rats; Staining and Labeling