Analysis of binding interaction between pegylated puerarin and bovine serum albumin by spectroscopic methods and dynamic light scattering

The interaction between bovine serum albumin (BSA) and pegylated puerarin (Pur) in aqueous solution was investigated by UV-Vis spectroscopy, fluorescence spectroscopy and circular dichroism spectra (CD), as well as dynamic light scattering (DLS). The fluorescence of BSA was strongly quenched by the binding of pegylated Pur to BSA. The binding constants and the number of binding sites of mPEG(5000)-Pur with BSA were 2.67±0.12 and 1.37±0.05 folds larger after pegylating, which were calculated from the data obtained from fluorescence quenching experiments. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be 4.09 kJ mol(-1) and 20.01 J mol(-1) K(-1), respectively, according to Van't Hoff equation, indicating that the hydrophobic force plays a main role in the binding interaction between pegylated Pur and BSA. In addition, the negative sign for Gibbs free energy change (ΔG) implies that the interaction process is spontaneous. Moreover, the results of synchronous fluorescence and CD spectra demonstrated that the microenvironment and the secondary conformation of BSA were changed. Comparing with Pur, all our data collected indicated that pegylated Pur interacted with BSA in the same way as that of Pur, but docked into the hydrophobic pocket of BSA with more accessibility and stronger binding force. DLS measurements showed monomethoxy polyethylene glycol (mPEG) have an effect on BSA conformation, and revealed that changes in BSA size might be due to increases in binding constant and the absolute values of ΔG after Pur pegylation.     

A review on the ligand binding studies by isothermal titration calorimetry

Thermodynamics of biomacromolecule ligand interaction is very important to understand the structure function relationship in proteins. One of the most powerful techniques useful to obtain additional information about the structure of proteins in biophysical chemistry field is isothermal titration calorimetry (ITC). An ITC experiment is a titration of a biomacromolecule solution by a solution containing a reactant (ligand) at constant temperature to obtain the exchanged heat of the reaction. The total concentration of ligand is the independent variable under experimental control. There are many reports on data analysis for ITC to find the number of binding sites (g), the equilibrium constant (K), the Gibbs free energy of binding process (ΔG), the enthalpy of binding (ΔH) and the entropy of binding (ΔS). Moreover, ITC gives information about the type of reaction, electrostatic and hydrophobic interactions, including determination of cooperativity characterization in binding process by calculating the Hill coefficient (n). A double reciprocal plot and a graphical fitting method are two simple methods used in the enzyme inhibition and metal binding to a protein. Determination of a binding isotherm needs more ITC experiments and more complex data analysis. Protein denaturation by ligand includes two processes of binding and denaturation so that ITC data analysis are more complex. However, the enthalpy of denaturation process obtained by ITC help to understand the fine structure of a protein.     

An isothermal titration calorimetry study of phytate binding to lysozyme

Isothermal titration calorimetry (ITC) was used to detect phytate binding to the protein lysozyme. This binding interaction was driven by electrostatic interaction between the positively charged protein and negatively charged phytate. When two phytate molecules bind to the protein, the charge on the protein is neutralised and no further binding occurs. The stoichiometry of binding provided evidence of phytate–lysozyme complex formation that was temperature dependent, being most extensive at lower temperatures. The initial stage of phytate binding to lysozyme was less exothermic than later injections and had a stoichiometry of 0.5 at 313 K, which was interpreted as phytate crosslinking two lysozyme molecules with corresponding water displacement. ITC could make a valuable in vitro assay to understanding binding interactions and complex formation that normally occur in the stomach of monogastric animals and the relevance of drinking water temperature on the extent of phytate–protein interaction. Interpretation of ITC data in terms of cooperativity is also discussed.     

Mechanism and conformational studies of farrerol binding to bovine serum albumin by spectroscopic methods

The mechanism and conformational changes of farrerol binding to bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence quenching technique, UV–vis absorption, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that farrerol could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The thermodynamic parameters enthalpy change and entropy change for the binding were calculated to be −29.92 kJ mol⁻¹ and 5.06 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which suggested that the both hydrophobic interactions and hydrogen bonds play major role in the binding of farrerol to BSA. The binding distance r deduced from the efficiency of energy transfer was 3.11 nm for farrerol–BSA system. The displacement experiments of site markers and the results of fluorescence anisotropy showed that warfarin and farrerol shared a common binding site I corresponding to the subdomain IIA of BSA. Furthermore, the studies of synchronous fluorescence, CD and FT-IR spectroscopy showed that the binding of farrerol to BSA induced conformational changes in BSA.     

The investigation of the interaction between NCP-EDA and bovine serum albumin by spectroscopic approaches

The fluorescence and ultraviolet spectroscopies were explored to study the interaction between N-confused porphyrins-edaravone diad (NCP-EDA) and bovine serum albumin (BSA) under simulative physiological condition at different temperatures. The experimental results show that the fluorescence quenching mechanism between NCP-EDA and BSA is a combined quenching (dynamic and static quenching). The binding constants, binding sites and the corresponding thermodynamic parameters (ΔG, ΔH, and ΔS) of the interaction system were calculated at different temperatures. According to F?rster non-radiation energy transfer theory, the binding distance between NCP-EDA and BSA was calculated to be 3.63 nm. In addition, the effect of NCP-EDA on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy.     

Study on the binding of luteolin to bovine serum albumin

Binding of luteolin (LU) to bovine serum albumin (BSA) was investigated at 298, 308 and 318K at pH 7.4 using spectrophotometric techniques such as fluorescence emission, circular dichroism (CD). The data obtained from fluorescence quenching experiments showed that LU was bound to BSA and binding constants and the number of binding sites (n approximately 1) were obtained. The thermodynamic parameters DeltaH(0), DeltaS(0), DeltaG(0) at different temperatures were calculated. They indicated that both hydrophobic forces and hydrogen bonds are the major interactions between LU and BSA. A value of 3.12nm for the average distance r between LU (acceptor) and tryptophan residue (Trp) of BSA (donor) was derived from the fluorescence resonance energy transfer. The effects of some common metal ions on the binding are also considered. Besides, the interaction of BSA with LU led to a change in the conformation of BSA.     

Hydration-dehydration of human serum albumin studied by isothermal calorimetry and IR spectroscopy

Based on a comparison of the data on the isothermal calorimetry of the interaction of human serum albumin with water and the adsorption isotherms of water vapor on the protein obtained by IR spectroscopy, an experimental method was used for the first time to study the thermochemical and sorption characteristics of protein hydration-dehydration over the entire range of the thermodynamic activities of water. A mechanism was proposed to explain the relationships between the thermochemical properties, protein water content, and the moistening method.     

Effect of pH on the interaction of vitamin B12 with bovine serum albumin by spectroscopic approaches

The interaction mechanism between vitamin B12 (B12, cyanocobalamin) and bovine serum albumin (BSA) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorbance, and three-dimensional fluorescence. The intrinsic fluorescence of BSA was strongly quenched by the addition of B12 in different pH buffer solutions (pH 2.5, 3.5, 5.0, 7.4, and 9.0) and spectroscopic observations are mainly rationalized in terms of a static quenching process at lower concentration of B12 (C(B12)/C(BSA)<5) and a combined quenching process at higher concentration of B12 (C(B12)/C(BSA)>5). The structural characteristics of B12 and BSA were probed, and their binding affinities were determined under different pH conditions. The results indicated that the binding abilities of B12 to BSA in the acidic and basic pH regions (pH 2.5, 3.5, 5.0, and 9.0) were lower than that at simulating physiological condition (pH 7.4). In addition, the efficiency of energy transfer from tryptophan fluorescence to B12 was found to depend on the binding distance r between the donor and acceptor calculated using F?rster's theory. The effect of B12 on the conformation of BSA was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results showed that the binding of B12 to BSA causes apparent change in the secondary and tertiary structures of BSA.     

Interaction between PAMAM 4.5 dendrimer, cadmium and bovine serum albumin: a study using equilibrium dialysis, isothermal titration calorimetry, zeta-potential and fluorescence

Binding of Cd²⁺ by PAMAM 4.5 dendrimer was studied by equilibrium dialysis, isothermal titration calorimetry and zeta-potential measurement. The following binding parameters were obtained: n = 23.8 ± 9.5, K_b = 4.7 ± 0.9 × 10³ in water; and n = 41.3 ± 13.4, K_b = 2.1 ± 0.8 × 10³ in 0.15 mol/l phosphate-buffered saline. The location of the bound Cd²⁺ is discussed. The interactions between bovine serum albumin, PAMAM 4.5 dendrimer and cadmium were analyzed using fluorescence and equilibrium dialysis. The competition between Cd²⁺ binding to BSA and PAMAM 4.5 dendrimer was investigated. It is proposed that PAMAM 4.5 dendrimer could be successfully used for extracting Cd²⁺ from aqueous solutions (environmental protection).     

Gadolinium and calcium binding to bovine serum albumin

The temperature jump relaxation technique is used to study the binding of calcium(II) and gadolinium(III) to bovine serum albumin at 7°C and an ionic strength of 0·2 (NaClO₄). Both metal ions are predicted to bind to the ϵ-amino groups of the protein above pH = 8·2, where extensive metal ion hydrolysis takes place. For the reaction between GdOH²⁺ and bovine serum albumin, the complex formation rate constant is (1·0±0·1) × 10⁸ M⁻¹ sec⁻¹ and the dissociation rate constant is (9·3±2·1) sec⁻¹. The corresponding calcium association reaction is too rapid to obtain rate constants. The complex formation rate constant is compared to other gadolinium systems to explain the relatively high complexation constant with bovine serum albumin.     

Application of a new method for data analysis of isothermal titration calorimetry in the interaction between human serum albumin and Ni

The interaction of human serum albumin (HAS) with divalent nickel ion was studied by isothermal titration calorimetry (ITC) in 30 mM Tris buffer, pH 7.0. There is a set of eight identical and independent binding sites for nickel ions on the protein at the temperature of 300 K. A new calorimetric data analysis allows the determination of the complete set of thermodynamic parameters. The binding isotherm for nickel-HSA interaction is easily obtained by carrying out two different ITC experiments. In the first experiment, the enthalpy of binding for one mole of nickel ion to one mole of binding site on HSA (ΔH=−36.5 kJ) is obtained, and is used in a second experiment to determine the binding isotherm and to find the number of binding sites (g=8) and the equilibrium constant (K=0.57 μM⁻¹).     

Study of caffeine binding to human serum albumin using optical spectroscopic methods

The binding of caffeine to human serum albumin (HSA) under physiological conditions has been studied by the methods of fluorescence, UV-vis absorbance and circular dichroism (CD) spectroscopy. The mechanism of quenching of HSA fluorescence by caffeine was shown to involve a dynamic quenching procedure. The number of binding sites n and apparent binding constant K _b were measured by the fluorescence quenching method and the thermodynamic parameters ΔH, ΔG, ΔS were calculated. The results indicate that the binding is mainly enthalpy-driven, with van der Waals interactions and hydrogen bonding playing major roles in the reaction. The distance r between donor (HSA) and acceptor (caffeine) was obtained according to the Förster theory of non-radiative energy transfer. Synchronous fluorescence, CD and three-dimensional fluorescence spectroscopy showed that the microenvironment and conformation of HSA were altered during the reaction.     

荧光光谱法研究左西孟旦与牛血清白蛋白的结合反应

用荧光光谱法、分光光度法研究了水溶液中左西孟旦与牛血清白蛋白(BSA)的相互结合反应。研究表明:左西孟旦对BSA的内源荧光有较强的猝灭作用且该猝灭作用属于静态荧光猝灭作用。得出了反应的结合常数(KA=1.48×106L/mol)和结合位点数(n=1.14)。根据Frster非辐射能量转移机理,求算了给体(BSA)与受体(左西孟旦)间距离r=2.9 nm和能量转移效率E=0.33。     

Study on the interaction between salvianic acid A sodium and bovine serum albumin by spectroscopic methods

The interaction between salvianic acid A sodium (SAS) and bovine serum albumin (BSA) was investigated using fluorescence and ultraviolet spectroscopy at different temperatures under imitated physiological conditions. The experimental results showed that the fluorescence of BSA was quenched by SAS through a static quenching procedure. The binding constants of SAS with BSA were 2.03, 1.17 and 0.71×10(5) L mol(-1) at 291, 298 and 305 K, respectively. Negative values of ΔG, ΔH, and ΔS indicate that the interaction between SAS and BSA is driven by hydrogen bonds and van der Waals forces. According to F?rster non-radiation energy transfer theory, the binding distance between BSA and SAS was calculated to be about 2.92 nm. The effect of SAS on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy. In addition, the effect of some metal ions Cu(2+), Ca(2+), Mg(2+), and Zn(2+) on the binding constant between SAS and BSA was examined.     

Effect of dioxane on the hydration of human serum albumin as studied by isothermal calorimetry and IR spectroscopy

A comparison of isothermal calorimetry data on the interaction of human serum albumin with water in the presence and absence of dioxane and of the isotherms of adsorption of vapors of water and dioxane on HSA as measured by IR spectroscopy made it possible to suggest an experimental method for isolating the contribution from the organic solvent to the thermodynamic and sorption characteristics of hydration of the protein over the entire range of water activities.     

Dissecting the cholera toxin-ganglioside GM1 interaction by isothermal titration calorimetry

The complex of cholera toxin and ganglioside GM1 is one of the highest affinity protein-carbohydrate interactions known. Herein, the GM1 pentasaccharide is dissected into smaller fragments to determine the contribution of each of the key monosaccharide residues to the overall binding affinity. Displacement isothermal titration calorimetry (ITC) has allowed the measurement of all of the key thermodynamic parameters for even the lowest affinity fragment ligands. Analysis of the standard free energy changes using Jencks' concept of intrinsic free energies reveals that the terminal galactose and sialic acid residues contribute 54% and 44% of the intrinsic binding energy, respectively, despite the latter ligand having little appreciable affinity for the toxin. This analysis also provides an estimate of 25.8 kJ mol(-1) for the loss of independent translational and rotational degrees of freedom on complexation and presents evidence for an alternative binding mode for ganglioside GM2. The high affinity and selectivity of the GM1-cholera toxin interaction originates principally from the conformational preorganization of the branched pentasaccharide rather than through the effect of cooperativity, which is also reinvestigated by ITC.     

Measurement of cation binding to immobilized vanillin by isothermal calorimetry

Isothermal calorimetry was used to determine enthalpy changes for interaction of divalent cobalt, nickel, copper, and zinc chlorides with silica gel functionalized with vanillin, Sil-Van. The thermal effect, Q(int), and the corresponding amount of cation that interacts, n(int), were obtained in the same experiment. Langmuir expressions for adsorption isotherms were applied to determine the maximum adsorption capacity to form a monolayer, N(mon), and the energy of interaction for a saturated monolayer per gram of Sil-Van, Q(mon). From knowledge of N(mon) and Q(mon), the molar enthalpy of interaction for formation of a monolayer of anchored cations per gram of Sil-Van, Delta(mon)H(m), was determined. Interactions between the Lewis-acidic cations and the donor atom attached to silica are reflected by Delta(mon)Hm values in the order Ni2+ > Cu2+ > Zn2+ congruent with Co2+.     

Studies of the interaction between apigenin and bovine serum albumin by spectroscopic methods

The interaction between apigenin (Ap) and bovine serum albumin (BSA) in physiological buffer (pH = 7.4) is investigated by fluorescence quenching technique and UV-vis absorption spectra. The results reveal that Ap could strongly quench the intrinsic fluorescence of BSA. The quenching mechanism of Ap for BSA varies with the change of Ap concentration. when Ap concentration is lower, it is a static quenching procedure, when Ap concentration is higher, a combined quenching (both static and dynamic) would operate. The apparent binding constants Ka and number of binding sites n of Ap with BSA are obtained by fluorescence quenching method. The thermodynamic parameters, enthalpy change (Δ_r H ^m and entropy change (Δ_r S ^m ), are calculated to be −15.382 kJ mol⁻¹ K⁻¹ < 0 and 104.888 J mol⁻¹ K⁻¹ > 0, respectively, which indicate that the interaction of Ap with BSA is driven mainly by hydrogen bonding and hydrophobic interactions. The distance r between BSA and Ap is calculated to be 1.89 nm based on F?rster’s non-radiative energy transfer theory. The results of synchronous fluorescence spectra show that binding of Ap with BSA cannot induce conformational changes in BSA.