Microcalorimetry, energetics and binding studies of DNA-dimethyltin dichloride complexes

The interaction of dimethyltin dichloride (Me₂SnCl₂) with calf thymus DNA was studied at 27 °C, pH 7.6 using various techniques including isothermal titration calorimetry (ITC) and UV-Vis, fluorescence and IR spectrophotometries. The binding isotherm and enthalpy curve for Me₂SnCl₂-DNA interaction was a biphasic transition process. This was determined by the analysis of the binding data with the Hill equation. The first phase of the enthalpy curve (exothermic process) was consistent with the first set of binding site, the second phase (endothermic process, less exothermicity) was consistent with second set of binding site from the cited interactions. Our results showed that the first set of binding sites is occupied by one mole of ligand bound per near 1 base pair of DNA. The DNA-ethidium bromide (EB) complex, in the presence of Me₂SnCl₂, caused the quenching of the fluorescence emission. The Scatchard plots illustrated a non-intercalating manner for such quenching. The DNA-EB complex results indicated that the binding of Me₂SnCl₂ is with the phosphate groups of DNA at low ligand concentrations (<9 mM). This was confirmed with the IR spectrophotometric spectra. However, the binding at higher ligand concentrations (>9 mM) was with the base groups of DNA. Therefore, these results suggest that the Me₂SnCl₂ binding to DNA at low concentrations occurs through an outside interaction by an exothermic process. However, the partial unfolding of the DNA caused at higher concentrations of Me₂SnCl₂ is through an endothermic process involving interactions with the base groups.     

Structural changes in β-lactoglobulin by conjugation with three different kinds of carboxymethyl cyclodextrins

β-Lactoglobulin-carboxymethyl cyclodextrin (β-LG-CMCyD) conjugates were prepared by using water soluble carbodiimide. Three kinds of CMCyDs differing in molecular mass were used to investigate the effects of different CMCyD contents, net charge and hydrophobicity on the structural changes in β-lactoglobulin. The effect of CMCyDs on the structure of β-lactoglobulin was utilized to investigate the contribution of hydrophobic interactions to the stability of the protein. Spectroscopic studies suggested that the conformation around had not changed in either conjugate but the α-helix content of β-LG-CMCyD conjugates had markedly increased as compared with that of β-lactoglobulin. The differential scanning calorimetry technique confirmed that the addition of one glucose unit in β-LG-CMCyD conjugates, enthalpy change of calorimetry decreased and the denaturation temperature of each conjugate was higher than that of native β-lactoglobulin. The heat contents agreed well with the conformational transition measured by molar ellipticity at 222 nm ([θ]₂₂₂) and Stoke's radius (R_S) values. Therefore, hydrophobic forces play an important role in stabilizing and shielding of the β-LG-CMCyD conjugates.     

A comparison of the inclusion behavior of human serum albumin and holo transferrin with fluoxymesterone in the presence of three different cyclodextrins

This article describes the interaction of fluoxymesterone (Flu) with HSA and HTF in the absence and presence of cyclodextrins (CDs) (α, β and γ). According to fluorescence data, the binding of Flu to the proteins caused strong static quenching in the binary and ternary systems. The fluorescence quenching results demonstrated that HSA and HTF had two and one class of apparent binding sites with a distinct binding constant in the presence of the CDs, respectively. The effects of Flu on the structure of HSA and HTF were analyzed using synchronous fluorescence spectroscopy, which showed that the interaction of Flu with both proteins in the binary and ternary systems altered the microenvironment around the Trp and Tyr residues. The distance, r, between Flu and the proteins was obtained according to FRET which pointed at a successful formation of a drug-protein complex. Far-UV CD spectra indicated that the binding of the drug to both proteins induced changes in the secondary structure of HSA and HTF in the binary and ternary systems. Finally, molecular modeling provided possible binding sites of Flu within the proteins for the binary and ternary systems and also confirmed the experimental results. The obtained data can be useful for determining usage drug doses in drug delivery.     

Theoretical study of the electronic conduction through organic nanowires

A substantial amount of researches have been carried out on the electron transport properties of gold surfaces. In order to study the role of linkage in the conductive properties of a molecular wire, different linkers such as sulfur, nitrogen, oxygen, CS, SH, NS, and CN are considered in our study. It is found that nitrogen or sulfur linkages can bond Au covalently to cis- and trans-butadiene, whereas on the other hand, oxygen linkage with the same shows a weak interaction and a non-covalent character. Further, this research is also an attempt to study the dependence of the molecular electronic structure of gold-molecule complexes on the external electric field. In addition, electronic conduction has been investigated from the perspective of alteration in shape of molecular orbitals and the development of the HOMO-LUMO gap of moleculegold complexes under the effect of an electric field.     

Multi-spectroscopic Investigations of Aspirin and Colchicine Interactions with Human Hemoglobin: Binary and Ternary Systems

The interactions of colchicine (COL) and aspirin (ASA) with human hemoglobin (HB) was studied by fluorescence, UV/vis absorption, resonance light scattering, synchronous fluorescence and circular dichroism (CD) spectroscopic techniques under physiological conditions. The inherent binding information, including the quenching mechanism, binding constants, number of binding sites, effective quenching constant, fraction of the initial fluorescence and thermodynamic parameters were determined by the fluorescence quenching technique at different temperatures. The results proved that the mechanism of fluorescence quenching of HB by COL and ASA is due to formation of HB–drug complexes in the binary and ternary systems. The distance between the acceptor drugs and HB was estimated by Förster’s equation on the basis of fluorescence energy transfer. In addition, according to the synchronous fluorescence spectra of HB, the results showed that the fluorescence quenching of HB originated solely from the tryptophan residues and indicated a conformational change for HB caused by addition of the drugs. Far-UV CD spectra of HB were recorded before and after the addition of ASA and COL both as binary and ternary systems. An increase in intensity of the positive CD peak of HB was observed in the presence of these drugs. The results were interpreted as excited state interactions between the aromatic residues of the HB binding sites and the drugs bound to them.     

Characterization of the interaction between human lactoferrin and lomefloxacin at physiological condition: Multi-spectroscopic and modeling description

The interaction between lomefloxacin (LMF) and human lactoferrin (Hlf) was studied by using fluorescence, circular dichroism (CD) spectroscopic and molecular modeling measurements. By the fluorescence quenching results, it was found that the binding constant K_A=8.69×10⁵ L mol⁻¹, and number of binding sites n=1.75 at physiological condition. Experimental results observed showed that the binding of LMF to Hlf induced conformational changes of Hlf. The participation of tyrosyl and tryptophanyl residues of protein was also estimated in the drug-Hlf complex by synchronous fluorescence. The quantitative analysis data of far-UV CD spectra from that of the α-helix 37.4% in free Hlf to 30.2% in the LMF-Hlf complex further confirmed that secondary structure of the protein was changed by LMF. Near-UV CD showed perturbations around tryptophan and tyrosine residues which involves perturbations of tertiary structure. The thermodynamic parameters like, ΔH° and ΔS°, have been calculated to be 63.411 kJ mol⁻¹ and 231.104 J mol⁻¹ K⁻¹, respectively. Thermodynamic analysis showed that hydrophobic interactions were the main force in the binding site but the hydrogen bonding and electrostatic interaction could not be excluded which in agreement with the result of molecular docking study. The distance r between donor and acceptor was obtained according to fluorescence resonance energy transfer (FRET) and found to be 1.78 nm. The interaction between LMF and Hlf has been verified as consistent with the static quenching procedure and the quenching mechanism is related to the energy transfer. Furthermore, the study of molecular modeling that LMF could bind to the α-helixes between Pro145-Asn152 and Phe167-Gln172 regions and hydrophobic interaction was the major acting force for the binding site, which was in agreement with the thermodynamic analysis.     

Magnetic solid-phase extraction of warfarin and gemfibrozil in biological samples using polydopamine-coated magnetic nanoparticles via core-shell nanostructure

Herein, polydopamine-coated Fe₃O₄ spheres were synthesized using a very simple, easy, cost-effective, efficient, and fast method. First, magnetic nanoparticles (Fe₃O₄) were synthesized and were followed by accommodating polydopamine on the surface of the prepared Fe₃O₄. The prepared polydopamine-coated Fe₃O₄ spheres were utilized as a sorbent in magnetic solid phase extraction of gemfibrozil and warfarin (as the model analytes). The extracted model analytes were desorbed by a suitable organic solvent and were analyzed by high-performance liquid chromatography. Under optimized condition, the linearity of the method was in the range of 0.1–200.0 μg/L for the selected analytes in water. The limits of detection were calculated to be in the range of 0.026–0.055 μg/L for warfarin and gemfibrozil, respectively. The limits of quantification were calculated to be in the range of 0.089–0.185 μg/L. The inter-day and intra-day relative standard deviations were determined to be in the range of 1.4%–3.3% in three concentrations in order to calculate the method precision. Furthermore, the enrichment factors were found to be 78 and 81 for warfarin and gemfibrozil, respectively. Moreover, the calculated absolute recoveries were between 78% and 81%. The obtained recoveries indicated that the method was useful and applicable in complicated real samples.     

Cooperative alpha-helix formation of beta-lactoglobulin induced by sodium n-alkyl sulfates

It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of beta-lactoglobulin was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at special condition. The effect of n-alkyl sulfates on the structure of native beta-lactoglobulin at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of non-native alpha-helical intermediate. The addition of various concentrations of n-alkyl sulfates to the native state of beta-lactoglobulin (pH 2) appears to support the stabilized form of non-native alpha-helical intermediate at pH 2. The m values of the intermediate state of beta-lactoglobulin by SOS, SDeS, SDS and STS showed substantial variation. The enhancement of m values as the stability criterion of non-native alpha-helical intermediate state corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the folding reaction of beta-lactoglobulin follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the non-native alpha-helical intermediate state.     

Study of the ropinirole hydrochloride interactions with human holo-transferrin in the presence of common metal ions

The binding of ropinirole hydrochloride (REQUIP) to human holo-transferrin (hTf) in the absence and presence of common ions has been investigated by fluorescence spectroscopy combined with fluorescence anisotropy, time-resolved fluorescence and circular dichroism (CD) under simulative physiological conditions. The quenching of the fluorescence intensity and the red shift in the maximum wavelength revealed an increased polarity of the microenvironment of the tryptophan and tyrosine residues. The number of binding sites and the apparent binding constants of REQUIP with hTf in the presence of Co²⁺, Fe³⁺, Cr³⁺, Al³⁺, Ca²⁺, Pb²⁺, Co ₃ ^2? , Cu²⁺, Mg²⁺ and K⁺ ions were determined. Time-resolved fluorescence decay profile gives the lifetime components reduce from 4.20 to 3.57?ns. Steady-state and time-resolved fluorescence data illustrated that the fluorescence quenching of complexes are static mechanism. Gradual addition of hTf led to marked increase in fluorescence anisotropy. From the value of anisotropy, it is argued that the REQUIP is located in a restricted environment of hTf. The quantitative analysis of CD spectra indicated that, in the presence of Co²⁺ and Fe³⁺ ions, the ??-helical structure content of hTf increased and for the other common ions, the ??-helical content decreased. In addition, thermodynamic analysis demonstrated that the van der Waals forces and hydrogen bond interactions stabilized the hTf?CREQUIP complex. These experimental results revealed that REQUIP could bind to hTf and those common ions, therefore could be a useful guideline for further therapeutic projects.     

Calorimetric evidence for conformational transitions of RNase A in the presence of cytidine 2′,3′-cyclic phosphate

Thermal denaturation of ribonuclease A (RNase A) complex with cytidine 3′-monophosphate (3′CMP) was studied by differential scanning calorimetry (DSC). The kinetic and binding studies of RNase A with cytidine 2′,3′-cyclic phosphate (cCMP) as a substrate, and 3′CMP as a ligand were also investigated by difference spectrophotometry. The obtained kinetic saturation curve reveals the occurrence of an anomalous non-hyperbolic shape at high substrate concentrations, and a biphasic binding isotherm. These phenomena indicate that a conformational change is occurring with RNase A during the hydrolysis of cCMP. A combination of kinetic and thermodynamic studies tends to elucidate the reasons for the formation of a non-hyperbolic behavior in a kinetic saturation curve. The thermal profile of the enzyme-3′CMP complexes shows a splitting of two distinct peaks with different structural stabilities of melting points (T_m) of 325 and 337 K. The bifurcate appearance of DSC profile of RNase A-3′CMP complexes manifests a physical view of a light kinetic structural transition. It is worthy to note, the direct binding (not via enzymatic reaction) of enzyme with 3′CMP indicates single DSC profile and monophasic binding isotherm.