Temperature dependence on the binding of butyl orange by bovine serum albumin

The binding of 4′-dibutylaminoazobenzene-4-sulfonate anion (butyl orange) by bovine serum albumin has been examined quantitatively by an equilibrium dialysis method at 5, 10, 15, 20, 25, and 35°C. The first binding constants and the thermodynamic parameters for the formation of the first dye anion-protein complex have been calculated. The peculiar temperature dependence of the first binding constant could be observed. That is, the value of the first binding constant increases with increasing temperature until it reaches a maximum value at approximately 18°C and then decreases with raising temperature. Accordingly, this binding process is exothermic above 18°C and is endothermic below 18°C. Near 18°C the process exhibits athermal reaction. From the thermodynamic data obtained, it is evident that the favorable free energy of the binding is accompanied by an entropy gain and that the enthalpies of the binding vary from a positive (unfavorable) value below 18°C to a negative (favorable) one above 18°C. Furthermore an apparent temperature dependence of the thermodynamic functions was observed. That is, ΔF° becomes larger in absolute magnitude as the temperature increases. The positive quantity of ΔS° tends to decrease with increasing temperature. All these facts can be interpreted satisfactorily in terms of hydrophobic interactions between hydrophobic portions of the dye and nonpolar parts of the albumin.     

Interaction of crosslinked polyvinylpyrrolidone with a homologous series of methyl orange derivatives in aqueous and nonaqueous solutions: Thermodynamics of the binding equilibria

The extent of binding of methvI orange, ethyl orange, propyl orange, and butyl orange by crosslinked polyvinylpyrrolidone was measured in all aqueous Solution. The first binding constants and the thermodynamic parameters accompanying the binding were evaluated. These values were compared with those of water-soluble polyvinylpyrrolidone. The first binding constant, the absolute magnitude of ΔF°, and the value of ΔS° of the crosslinked polyvinylpyrrolidone are substantially larger than those of the water-soluble product for any particular dye. These behaviors can be accounted for in terms of increased hydrophobic domains in the former and enhanced hydrophobic contribution in the binding process. Also the binding of the dye by the crosslinked polymer in a nonaqueous solvent, ethylene glycol, was measured to assess the contribution of hydrophobic interaction to the dye-polymer complex formation in aqueous medium. It was found that the binding of butyl orange by the crosslinked polymer is suppressed in ethylene glycol and the contribution of entropy term to the free energy change in the aqueous environment is large compared with that in ethylene glycol. The significance of the hydrophobic of the hydrophobic interaction in the dye-polymer association process is described.     

Thermodynamics of binding of methyl orange and its homologs by poly(vinylpyrrolidone): The effect of inorganic electrolytes

The extent of binding of methyl orange, ethyl orange, propyl orange, and butyl orange by poly(vinylpyrrolidone) has been measured in aqueous solutions of inorganic electrolytes such as NaCl, LiCl, NaSCN, and NaClO₄ by an equilibrium dialysis method. The effect of the salts on the first binding constants and the thermodynamic functions which are accompanied by the dye—polymer association process was investigated relative to the corresponding values in the absence of such salts. It was found that in aqueous solutions of NaCl and LiCl the enthalpy change accompanying the binding is small and the largest contribution to the free energy of binding is from the positive entropy gain. For NaSCN and NaClO₄, the values of ΔF° and ΔH° were both large and negative and the value of ΔS° was small and negative. Thus, the favorable free energy for the complex formation was due entirely to the negative enthalpy term. These characteristics of the thermodynamic quantities are discussed in terms of changes in structural properties of water in the vicinity of the binding entities and conformational changes of the polymer to which the dye is bound due to the added foreign electrolytes.     

Binding of methyl orange and its homologs by powdered nylon 66

Powdered Nylon 66 was prepared as a model of amorphous polymers. The resultant powder polyamide was composed of only amorphous regions. The extent of uptake of the acid azo dyes, a homologous series of methyl orange derivatives, by the polymer was measured in an aqueous solution. The first binding constants and the thermodynamic parameters in the course of the binding were evaluated. The thermodynamic behaviors obtained are very similar to those of crosslinked polyvinylpyrrolidone. The favorable free energy of the binding is accompanied by an entropy gain and an exothermic enthalpy change. The shorter the alkyl chain of the dyes, the more negative is the enthalpy change and, hence, the smaller is the entropy change. The thermodynamic data for butyl orange showed that the binding process is athermal and is wholly an entropic effect. The binding of the dyes to the matrix is entropically favorable as a result of the operation of the hydrophobic effect. In addition, an electrostatic force is operative between the sulfonate group on the dyes and the terminal amino groups on the polyamide.     

Interaction of polyvinylpyrrolidone with methyl orange and its homologs in aqueous solution: Thermodynamics of the binding equilibria and their temperature dependences

The interaction of polyvinylpyrrolidone with methyl orange, ethyl orange, propyl orange, and butyl orange has been studied by an equilibrium dialysis method at 5, 15, 25, and 35°C. The first binding constants and the thermodynamic parameters in the course of the binding have been calculated. It was found that the free energy and the enthalpy changes are all negative and the entropy change is largely positive. The longer the alkyl chain of the dyes, the more positive is the enthalpy change (though it is always in the negative direction) and hence the larger is the entropy change. The favorable free energy of the binding of butyl orange observed for the formation of the dye–polymer complex seems to be a result of a favorable entropy change rather than any favorable enthalpy change. Temperature dependences of the thermodynamic functions were apparently observed. That is, ΔF and ΔH become larger in absolute magnitude as the temperature increases. The positive quantity of ΔS tends to decrease with increasing temperture. All these facts obtained can be interpreted satisfactorily by the hydrophobic interaction between hydrocarbon portions of the dyes and nonpolar parts of the macromolecule.     

Thermodynamic contributions of the ordered water molecule in HIV-1 protease

Binding between biomolecules is usually accompanied by the formation of direct interactions with displacement of water from the binding sites. In some cases, however, the interactions are mediated by ordered water molecules, whose effect on binding affinity and the other thermodynamic functions is unclear. In this work, we compute the contribution of one such water molecule, the strongly bound water molecule at the binding site of HIV-1 protease, to the thermodynamic properties using statistical mechanical formulas for the energy and entropy. The requisite correlation functions are obtained by molecular dynamics simulations. We find that the entropic penalty of ordering is large but is outweighed by the favorable water-protein interactions. We also find a large negative contribution from this water molecule to the heat capacity. This approach could be useful in rational drug design by estimating which bound water molecules would be most favorable to displace.     

Spectral and thermodynamic studies of the interaction of binding site probes with poly(vinylpyrrolidone)

The binding of two ionic azo dyes (4-phenylazo-1-naphthol mono-and disulfonate) and a fluorescent probe (2-p-toluidinonaphthalene-6-sulfonic acid, TNS) to poly(vinylpyrrolidone) (PVP) was studied to obtain information on the nature of the interaction, binding isotherm, and binding site. Sorption of the dyes followed a Langmuir isotherm only at low polymer saturation. Apparent cooperativity in binding was seen at higher saturation. The polymer had a higher intrinsic binding constant but lower binding capacity for the doubly charged dye than for the structurally similar singly charged dye. Both dyes consisted of tautomeric mixtures of hydrazone and azonaphthol forms in equilibrium in the bound and unbound state. The preferential binding of the azonaphthol tautomer of the disulfonate was highly exothermic and accompanied by an entropy decrease. The binding of the hydrazone form was less favored by 1.8 kcal/mol, was weakly exothermic, and accompained by an entropy increase. Increased preference for the azonaphthol tautomer accompanied chain extension from charging the polymer. Chain extension had no effect on the emission frequency of bound TNS. Large differences in binding capacities for similarly charged dyes indicated the existence of specific dye-site interactions. Arguments are presented against nonspecific hydrophobic interactions as predominant forces responsible for binding.     

Temperature dependence on the binding of butyl orange by bovine serum albumin: The effect of urea

The effect of urea on the extent of the binding of butyl orange by bovine serum albumin has been examined by an equilibrium dialysis method. The first binding constants and the thermodynamic parameters for the formation of the first dye anion–protein complex have been calculated. Addition of urea to the binding system causes a marked decrease in the absolute magnitude of the free energy change. The enthalpy change during binding becomes more exothermic, and the entropy change tends to decrease with increasing concentration of urea. These results can be interpreted in terms of the concept that urea reduces the structure of the aqueous environment and hence lowers the tendency of apolar groups of the dye and the albumin to participate in the formation of hydrophobic interactions.     

Interaction of crosslinked polyethylenimine with a homologous series of methyl orange derivatives in aqueous solution

Polyethylenimine (PEI) was crosslinked with dichloroethane, glyoxal, or glutaraldehyde and polymers of various degrees of crosslinkage were made. The insoluble polymers obtained were examined for their ability to bind methyl orange and its homologs, methyl, ethyl, propyl, and butyl orange at 5, 15, 25, and 35°C, respectively, in an aqueous solution. PEI crosslinked with glutaraldehyde showed markedly increased binding affinity toward these cosolutes compared with the polymers crosslinked with dichloroethane or glyoxal. The extent of the binding increased with an increase in the degree of crosslinkage. These results suggest that the enhancement of the binding by the crosslinking is due mainly to a dual effect, introduction of hydrophobic moieties and proximity of neighboring polymer chains. The first binding constants and the thermodynamic parameters that accompanied the binding were calculated. The thermodynamic data show that the binding process is athermal and is stabilized entirely by the entropy term. Water-soluble PEI exhibited stronger cooperative interactions than the crosslinked polymer because the mobilities of the chains of the former are greater than those of the latter.     

Involvement of water in carbohydrate-protein binding.

The interactions of trimannosides 1 and 2 with Con A were studied to reveal the effects of displacement of well-ordered water molecules on the thermodynamic parameters of protein-ligand complexation. Trisaccharide 2 is a derivative of 1, in which the hydroxyl at C-2 of the central mannose unit is replaced by a hydroxyethyl moiety. Upon binding, this moiety displaces a conserved water molecule present in the Con A binding site. Structural studies by NMR spectroscopy and MD simulations showed that the two compounds have very similar solution conformational properties. MD simulations of the complexes of Con A with 1 and 2 demonstrated that the hydroxyethyl side chain of 2 can establish the same hydrogen bonds in a low energy conformation with the protein binding site as those mediated by the water molecule in the complex of 1 with Con A. Isothermal titration microcalorimetry (ITC) measurements showed that 2 has a more favorable entropy of binding compared to 1. This term, which was expected, arises from the return of the highly ordered water molecule to bulk solution. The favorable entropy term was, however, offset by a relatively large unfavorable enthalpy term. This observation was rationalized by comparing the extent of hydrogen bond and solvation changes during binding. It is proposed that an indirect interaction through a water molecule will provide a larger number of hydrogen bonds in the complex that have higher occupancies than in bulk solution, thereby stabilizing the complex.     

Binding methyl orange and hydrophobic fluorescent probes by poly(2-dimethylaminoethyl methacrylate) and copolymers of 2-dimethylaminoethyl methacrylate and N-vinyl-2-pyrrolidone: pH dependence of the binding and fluorescence intensity

The pH dependence of the interaction of poly(2-dimethylaminoethyl methacrylate) and copolymers of 2-dimethylaminoethyl methacrylate and N-vinyl-2-pyrrolidone with methyl orange, 2-p-toluidinylnaphthalene-6-sulfonate (TNS), and 1,6-diphenyl-1,3,5-hexatriene (DHT) was studied by equilibrium dialysis and fluorescence measurements at pH's 7–10. The first binding constant accompanying the binding of methyl orange and TNS by the polymers, in particular the homopolymer, shows a maximum around pH 8 and maximal fluorescence intensity of TNS is obtained around pH 8.5 in the presence of the polymers. To elucidate these observations the pH-induced conformational changes of the homopolymer were examined by potentiometric titration and viscosity measurements and the thermodynamic parameters that accompany the binding were calculated. The polymer was found to change from an extended coil at lower pH to a compact coil at higher pH. The electrostatic attraction between the sulfonate group of the small molecule and the protonated nitrogen atoms on the polymer is increased at lower pH and the hydrophobic interaction between the hydrophobic moieties of the polymer and the small molecule is enhanced at higher pH. The results obtained for the dye binding and fluorescence intensity were discussed in terms of the electrostatic and hydrophobic interactions.     

Structure and properties of the aluminum borates Al(BO2)n and Al(BO2)n(-), (n = 1-4)

The geometrical and electronic structures of Al(BO(2))(n) and Al(BO(2))(n)(-) (n = 1-4) clusters are computed at different levels of theory including density functional theory (DFT), hybrid DFT, double-hybrid DFT, and second-order perturbation theory. All aluminum borates are found to be quite stable toward the BO(2) and BO(2)(-) loss in the neutral and anion series, respectively. Al(BO(2))(4) belongs to the class of hyperhalogens composed of smaller superhalogens, and should possess a large adiabatic electron affinity (EA(ad)) larger than that of its superhalogen building block BO(2). Indeed, the aluminum tetraborate possesses the EA(ad) of 5.6 eV, which, however, is smaller than the EA(ad) of 7.8 eV of the AlF(4) supehalogen despite BO(2) is more electronegative than F. The EA(ad) decrease in Al(BO(2))(4) is due to the higher thermodynamic stability of Al(BO(2))(4) compared to that of AlF(4). Because of its high EA and thermodynamic stability, Al(BO(2))(4) should be capable of forming salts with electropositive counter ions. We optimized KAl(BO(2))(4) as corresponding to a unit cell of a hypothetical KAl(BO(2))(4) salt and found that specific energy and energy density of such a salt are competitive with those of trinitrotoluol (TNT).     

Binding the hydrophobic fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate,by polycations that contain apolar pendant groups: Equilibrium dialysis and fluorescence measurements

The thermodynamic parameters for the interaction of the hydrophobic fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS), and polycations that contain a piperidinium cation and various nonpolar pendant groups were calculated. Binding is exothermic and involves a positive entropy gain. The contribution of the entropy term to the free energy change tends to increase with increasing hydrophobicity of the polymers. The intensity of the fluorescence of TNS is enhanced when the probe binds to the polycations. The nature and phenomena of hydrophobic fluorescent probe binding with the polymers are discussed.     

Microcalorimetry and spectroscopic studies on the binding of dye janus green blue to deoxyribonucleic acid

The interaction of the phenazinium dye janus green blue (JGB) with deoxyribonucleic acid was investigated using isothermal titration calorimetry and thermal melting experiments. The calorimetric data were supplemented by spectroscopic studies. Calorimetry results suggested the binding affinity of the dye to DNA to be of the order of 10⁵ M^?1. The binding was predominantly entropy driven with a small negative favorable enthalpy contribution to the standard molar Gibbs energy change. The binding became weaker as the temperature and salt concentration was raised. The temperature dependence of the standard molar enthalpy changes yielded negative values of standard molar heat capacity change for the complexation revealing substantial hydrophobic contribution in the DNA binding. An enthalpy–entropy compensation behavior was also observed in the system. The salt dependence of the binding yielded the release of 0.69 number of cations on binding of each dye molecule. The non-polyelectrolytic contribution was found to be the predominant force in the binding interaction. Thermal melting studies revealed that the DNA helix was stabilized against denaturation by the dye. The binding was also characterized by absorbance, resonance light scattering and circular dichroism spectral measurements. The binding constants from the spectral results were close to those obtained from the calorimetric data. The energetic aspects of the interaction of the dye JGB to double-stranded DNA are supported by strong binding revealed from the spectral data.     

Molecular recognition in imprinted polymers: thermodynamic investigation of analyte binding using microcalorimetry

This study aimed at elucidating the interaction mechanism between an imprinted polymer and its template in aqueous environment with thermodynamic aspects. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was chosen as a model template to imprint a co-polymer of 4-vinylpyridine (4-VP) and ethyleneglycol dimethacrylate. Equilibrium binding isotherm analysis and isothermal titration microcalorimetry were used to quantify the contribution of enthalpy and entropy to the binding process, identify the nature of the interactions involved and confirm the existence of binding pockets with shape-complementarity to the template. For the binding process of 2,4-D to the imprinted polymer, we postulate three subprocesses: (1) dehydration of the binding pocket and of the 2,4-D, (2) adsorption of 2,4-D, and (3) rearrangement of the water molecules from the dehydration process. We found that binding in aqueous environment was due to the cumulative effect of pi-stacking and electrostatic interactions between the template and the functional monomers. At pH<6, entropy is the dominating driving force, while at pH>6 where the highest difference in binding between the imprinted and a non-imprinted reference polymer was observed, the enthalpy change accounts for most of the binding free energy. The developed microcalorimetric method sheds light on the binding mechanism of analyte molecules with imprinted polymers, in particular if the polymers are used in aqueous solvents.     

Thermodynamic and structural study of pyrene-1-carboxaldehyde/DNA interactions by molecular spectroscopy: Probing intercalation and binding properties

The binding of pyrene-1-carboxaldehyde (1-PyCHO) with ctDNA was investigated through absorption, intrinsic and induced circular dichroism, viscosity measurements and steady-state fluorescence. The binding and the number of monomer units of the polymer involved in the binding of one dye molecule (site size) have been quantified. The results indicated that the 1-PyCHO molecule binds to the ctDNA in an intercalative mode. The spectroscopic evidence of this intercalation process is also corroborated by the effect of urea, iodide-induced fluorescence quenching of pyrene-1-carboxaldehyde and competitive binding using a fluorescent intercalator, SYBR Green I (SG). The induced circular dichroism (ICD) spectra of pyrene-1-carboxaldehyde complexed with ctDNA show that pyrene-1-carboxaldehyde intercalates into ctDNA and that the intercalation orientation of pyrene to the DNA base-pairs long axis is heterogeneous. On the other hand, the intrinsic circular dichroism (CD) spectra show a stabilization of the right-handed B form of ctDNA, due to the intercalation process.     

含喹唑啉酮的4-(4-氟苯基)哌嗪二硫代甲酸酯与牛血清白蛋白相互作用的荧光光谱研究

用荧光光谱法研究了具有抑制人肿瘤细胞活性的含喹唑啉酮的4-(4-氟苯基)哌嗪二硫代甲酸酯与牛血清白蛋白的相互作用。结果表明:在生理条件下,它对牛血清白蛋白的荧光有较强的猝灭作用。根据猝灭结果,求得了不同温度下反应的结合位点数、结合常数及反应热力学参数,并据此确定了它们相互作用的主要形式。     

A model of cooperativity based on restricted binding: kinetic and thermodynamic analysis

Cooperative protein–ligand binding is an essential biochemical process. In this work, we introduce a model that can simulate the emergence of such phenomenon in the binding kinetics. It is based on the inability of the ligand molecules to fully utilize all the available binding sites due to some restriction, realized here in terms of a model parameter, called the restriction parameter. The theory is developed at the level of a single oligomeric protein molecule interacting with a ligand, maintained at a constant concentration, using a chemical master equation. The model provides stepwise binding constants related to the restriction parameter. The relative magnitudes of these constants, when compared to the Hill coefficients measuring cooperativity, give a physical insight in the development of the cooperative behavior and can also act as a reference frame. This can be useful for an alternative theoretical characterization of cooperativity in oligomeric proteins with large number of binding sites and arbitrary binding constants. We establish this point here by taking a tetrameric protein as a case study. A stochastic thermodynamic analysis is also performed, highlighting the energy–entropy contribution to the overall free energy change due to protein–ligand interaction for various cases of restricted binding.     

Conformational stabilization of an engineered binding protein

We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA)(-)(1) affibody binds to its Z domain binding partner with a dissociation constant K(d) = 1.6 microM, and previous analyses suggested that the moderate affinity is due to the conformational heterogeneity of free Z(SPA)(-)(1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA)(-)(1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K(d) = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA)(-)(1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA)(-)(1). Instead, higher Z domain binding affinity in a group of eight Z(SPA)(-)(1) variants correlates with more favorable binding enthalpy and enthalpy-entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.     

Energetics of phosphate binding to ammonium and guanidinium containing metallo-receptors in water

The design and synthesis of receptors containing a Cu(II) binding site with appended ammonium groups (1) and guanidinium groups (2), along with thermodynamics analyses of anion binding, are reported. Both receptors 1 and 2 show high affinities (10(4) M(-1)) and selectivities for phosphate over other anions in 98:2 water:methanol at biological pH. The binding of the host-guest pairs is proposed to proceed through ion-pairing interactions between the charged functional groups on both the host and the guest. The affinities and selectivities for oxyanions were determined using UV/vis titration techniques. Additionally, thermodynamic investigations indicate that the 1:phosphate complex is primarily entropy driven, while the 2:phosphate complex displays both favorable enthalpy and entropy changes. The thermodynamic data for binding provide a picture of the roles of the host, guest, counterions, and solvent. The difference in the entropy and enthalpy driving forces for the ammonium and guanidinium containing hosts are postulated to derive primarily from differences in the solvation shell of these two groups.