Interaction of wogonin with bovine serum albumin

The binding of wogonin with bovine serum albumin (BSA) was investigated at different temperatures by fluorescence, circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR) at pH7.40. The association constants K were determined by Stern-Volmer equation based on the quenching of the fluorescence of BSA in the presence of wogonin, which were in agreement with the constants calculated by Scatchard plots. The thermodynamic parameters were calculated according to the Van't Hoff equation and the result indicated that DeltaH(0) and DeltaS(0) had a negative value (-12.02 kJ/mol) and a positive value (58.72 J/mol K), respectively. On the basis of the displacement experimental and the thermodynamic results, it is considered that wogonin binds to site I (subdomain IIA) of BSA mainly by hydrophobic interaction. The studied results by FT-IR and CD experiment indicated that the secondary structures of protein have been perturbed by the interaction of wogonin with BSA.     

Binding of the bioactive component jatrorrhizine to human serum albumin

The interaction between Jatrorrhizine with human serum albumin (HSA) were studied by fluorescence quenching technique, circular dichroism (CD) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants (K) are 7.278 x 10(4), 6.526 x 10(4), and 5.965 x 10(4) L.mol(-1) at 296, 303, and 310 K, respectively. The CD spectra and FT-IR spectra have proved that the protein secondary structure changed in the presence of Jatrorrhizine in aqueous solution. The effect of common ions on the binding constants was also investigated. In addition, the thermodynamic functions standard enthalpy (DeltaH(0)) and standard entropy (DeltaS(0)) for the reaction were calculated to be -10.891 kJ.mol(-1) and 56.267 J.mol(-1) K(-1), according to the van't Hoff equation. These data indicated that hydrophobic and electrostatic interactions played a major role in the binding of Jatrorrhizine to HSA. Furthermore, the displacement experiments indicated that Jatrorrhizine could bind to the site I of HSA, which was also in agreement with the result of the molecular modeling study.     

Interaction of rhein with human serum albumin investigation by optical spectroscopic technique and modeling studies

The binding of rhein with human serum albumin (HSA) has been studied in detail by spectroscopic method including circular dichroism (CD), Fourier transformation infrared spectra (FT-IR), fluorescence spectra. The binding parameters for the reaction have been calculated according to Scatchard equation at different temperatures. The plots indicated that the binding of HSA to rhein at 303, 310 and 318 K is characterized by one binding site with the affinity constant K at (4.93+/-0.16)x10(5), (4.02+/-0.16)x10(5) and (2.69+/-0.16)x10(5) M-1, respectively. The secondary structure compositions of free HSA and its rhein complexes were estimated by the FT-IR spectra. FT-IR and curve-fitted results of amide I band are in good agreement with the analyses of CD spectra. Molecular Modeling method was used to calculate the interaction modes between the drug and HSA.     

Biochemical evaluation of a synthesized isoflavone-selenium complex by molecular spectra

A coordination compound of 5, 7-dihydrox-4'-methoxyisoflavone and selenium was synthesized and its structure was identified by IR, LC-MS and (1)H-NMR. Its biochemical effects were investigated using bovine serum albumin (BSA) as a target protein molecule, in which process three-dimensional (3D) fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra and fluorescence probe techniques were employed. The interaction of SEIF and BSA was discussed by fluorescence quenching method and F?rster non-radiation energy transfer theory. The thermodynamic parameters ΔH (θ), ΔG (θ), ΔS (θ) at different temperatures were calculated according to Van't Hoff isobaric equation and the results indicated the interaction was an exothermic as well as a spontaneous process. The binding site was explored by fluorescence probe method using warfarin and ibuprofen as markers. Intramolecular forces which are responsible for maintaining the binding were mainly hydrogen bond and van der Waals power. The average distance from the tryptophan residue in domain II of BSA (donor) to SEIF (acceptor) is 3.57 nm at body temperature. The conformation changes of BSA were investigated by 3D fluorescence and CD spectra.     

Binding of isofraxidin to bovine serum albumin

The binding of isofraxidin to bovine serum albumin (BSA) was studied under physiological conditions with BSA concentration of 1.5 x 10(-6) mol x L(-1) and drug concentration in the range of 1.67 x 10(-6) mol x L(-1) to 2.0 x 10(-5) mol x L(-1). Fluorescence quenching spectra in combination with uv absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and CD spectroscopy was used to determine the drug-binding mode, binding constant, and the protein structure changes in the presence of isofraxidin in aqueous solution. The linearity of Scatchard plot indicates that isofraxidin binds to a single class of binding sites on BSA and the values given for the binding constants agree very closely with those obtained by the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated to be -17.63 kJ x mol(-1) and 51.38 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to BSA.     

Study on the binding of Arsenazo-TB to human serum albumin by Rayleigh light scattering technique and FT-IR

The interaction between Arsenazo-TB and human serum albumin (HSA) was studied by Rayleigh light scattering (RLS) technique and Fourier transformed IR (FT-IR). The binding parameters of Arsenazo-TB with HSA were studied at different temperature of 288, 298, 308, 318 K under the optimum conditions. It is indicated by the Scatchard plots that the binding constant K decreased from 5.03 x 10(7) to 7.13 x 10(6) and the maximum binding number N reduced from 53 to 36 with the increasing of the temperature. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction. The free energy change deltaG0, the enthalpy change deltaH0 and the entropy change deltaS0 of 288 K were calculated to be -42.46 kJ/mol, -49.17 kJ/mol and 318.15 J/mol K, respectively. The alterations of protein secondary structure in the presence of Arsenazo-TB in aqueous solution were quantitatively calculated from FT-IR spectroscopy with reductions of alpha-helix from 57% to 40% and with increases of beta-sheet from 36% to 39%, beta-turn from 7% to 21%.     

Interaction of pirprofen enantiomers with human serum albumin.

The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule.     

Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies

BACKGROUND: Fluorescent markers (labeled antibodies) and flow cytometry are used to enumerate the average number of receptors (antigens) on formed bodies (cells) in whole blood by using a new method that avoids the extra steps of separating bound from unbound fluorescent markers or the use of external standards. METHODS: Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by standard cytometry procedures are used to complete the analyses for receptors per cell. Also, flow cytometric assays using competitive binding between fluorescent marker (CD4-RD1, CD8-FITC, CD3-FITC, CD3-RD1) and unlabeled antibody (CD4, CD8, CD3, CD3-dextran) for receptors on white blood cells in whole blood are described for determination of relative and specific binding constants of unlabeled/labeled antibody for targeted receptors. RESULTS: Ranges that were obtained for receptors per cell (lymphocytes) in normal blood donors were as follows: CD4, 4.9 x 10(4)-1.5 x 10(5); CD8, 5.0 x 10(5)-2.1 x 10(6); CD3, 6.6-7.8 x 10(5). Binding constants were highest for unlabeled CD4 antibody, 2. 7 x 10(10)-2.1 x 10(12) M(-1), and then unlabeled CD3 antibody, 1.1 x 10(10)-1.9 x 10(11) M(-1). FITC- and RD1-labeled antibodies typically had binding constants that were 10-to 100-fold lower than the native antibodies. CONCLUSIONS: Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.     

Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate. No protection from thrombin digestion was afforded by tRNA(gly). Binding constants were determined by isothermal titration calorimetry at 25 degrees C for ATP (2.5 x 10(5) M(-1)), AMPPNP (3.7 x 10(5) M(-1)), and AMPPCP (2.2 x 10(6) M(-1)). The nucleotides had similar values for DeltaH (-71 kJ mol(-1)), with values for TDeltaS that accounted for the differences in the binding constants. Near-ultraviolet CD spectra of the enzyme-nucleotide complexes indicate that the nucleotides are bound in the anti configuration. A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for DeltaH (-187 kJ mol(-1)), which was balanced by a large TDeltaS term to give a binding constant (3.7 x 10(6) M(-1)) only slightly larger than that of AMPPCP. Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP. AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase. Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (-2.6 kJ K(-1) mol(-1)). Titrations performed in buffers with different ionization enthalpies indicate that the large value for DeltaH for the adenylate analogue does not arise from a coupled protonation event. Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides. Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined. The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.     

Characterization of the interaction between reserpine and bovine serum albumin: spectroscopic approaches

The characteristics of the interaction between reserpine and bovine serum albumin (BSA) were studied by fluorescence, UV-vis absorption and Fourier transform infrared (FT-IR) spectroscopy. Spectroscopic analysis revealed that fluorescence quenching of BSA by reserpine was through a static quenching procedure. The binding constant K(A) of reserpine with BSA at 293, 301 and 309 K was 1.63, 1.78 and 2.35 x 10(5) moL(-1) L respectively, which indicated degree of binding force between reserpine and BSA. There was one binding site between reserpine and BSA. The entropy and enthalpy changes were positive, indicating that interaction of reserpine and BSA was driven mainly by hydrophobic forces. The average binding distance between the donor (BSA) and the acceptor (reserpine) was about 3.84 nm based on the Forster non-radiation energy transfer theory. Results of synchronous fluorescence and FT-IR spectra indicated that the conformation and microenvironment of BSA were changed by the binding of reserpine. The results may provide important insights into the physiological activity of reserpine.     

Dissection of binding of trypsin to its natural inhibitor Gensenoside-Rg1 using spectroscopic methods and molecular modeling

Abstract

The interaction of trypsin with Gensenoside-Rg1 (G-Rg1) was studied using fluorescence, ultraviolet–visible (UV–vis), and circular dichroism (CD) spectroscopies along with enzyme activity assay and molecular docking. The enzyme activity assays showed that G-Rg1 inhibited the activity of trypsin effectively. The fluorescence experiments indicated that a complex of G-Rg1–trypsin was formed and that the fluorescence of trypsin was quenched by G-Rg1 via a mixed-quenching mechanism (both static and dynamic quenching). The thermodynamic analysis suggested that hydrophobic interaction and hydrogen bond were the major forces between G-Rg1 and trypsin. According to the theory of Förster’s non-radiation energy transfer, the binding distance between trypsin and G-Rg1 was calculated to be 2.01?nm, which implies that energy transfer occurred within the complex. The experimental results obtained from UV–vis absorption spectra, synchronous fluorescence spectra, and CD spectra indicated that G-Rg1 was mainly located on tryptophan moiety and that the interaction between G-Rg1 and trypsin led to conformational changes of trypsin with some α-helix and unordered coil structures being transformed into β-sheet structures. In addition, docking results supported the above experimental findings and suggested the possible binding location of G-Rg1 on trypsin along with the possible hydrogen bonds and hydrophobic interactions between G-Rg1 and trypsin. The experimental results from this study should be useful to minimize the antinutritional effects and make full use of Genseng extracts in the food industry and also be helpful to the design of the drugs for the diseases related to overexpression of trypsin.

Communicated by Ramaswamy H. Sarma     

Elucidation of the mechanism of interaction of sheep spleen galectin-1 with splenocytes and its role in cell-matrix adhesion

The binding of a 14 kDa beta-galactoside animal lectin to splenocytes has been studied in detail. The binding data show that there are two classes of binding sites on the cells for the lectin: a high-affinity site with a K(a) ranging from 1.1 x 10(6) to 5.1 x 10(5) M (-1) and a low affinity binding site with a K(a) ranging from 7.7 x 10(4) to 3.4 x 10(4) M (-1). The number of receptors per cell for the high- and low-affinity sites is 9 +/- 3 x 10(6) and 2.5 +/- 0.5 x 10(6), respectively. The temperature dependence of the K value yielded the thermodynamic parameters. The energetics of this interaction shows that, although this interaction is essentially enthalpically driven (DeltaH - 21 kJ lambdamol(-1)) for the high-affinity sites, there is a very favorable entropy contribution to the free energy of this interaction (-TDeltaS - 17.5 Jmol(-1)), suggesting that hydrophobic interaction may also be playing a role in this interaction. Lactose brought about a 20% inhibition of this interaction, whereas the glycoprotein asialofetuin brought about a 75% inhibition, suggesting that complex carbohydrate structures are involved in the binding of galectin-1 to splenocytes. Galectin-1 also mediated the binding and adhesion of splenocytes to the extracellular matrix glycoprotein laminin, suggesting a role for it in cell-matrix interactions.     

Fluorometric probing on the binding of hematoxylin to serum albumin

The binding of a cell nucleus stain, hematoxylin (HTL), to bovine serum albumin (BSA) was studied by spectroscopy including fluorescence spectra, UV–Visible absorption, circular dichroism (CD) spectra, synchronous and three-dimensional fluorescence spectra. The results indicated that the binding had led to static fluorescence quenching, with non-radiation energy transfer happening within single molecule. The observed binding constant was calculated to be 10^5.588 l mol^?1 at 311 K and one binding site had formed. The thermodynamic parameters of the interaction complied with ΔG ^θ < 0, ΔH ^θ < 0, ΔS ^θ < 0 and the results indicate that hydrogen bonds played major role in the reaction. The distance r between donor (BSA) and acceptor (HTL) was obtained according to the Förster theory of non-radiation energy transfer. The structural change of BSA molecules with addition of HTL was analyzed and the optimized geometry of HTL–BSA was investigated by fluorescence probe method.     

Comparison and analysis on the serum‐binding characteristics of aspirin–zinc complex and aspirin

This study was designed to compare the protein‐binding characteristics of aspirin–zinc complex (AZN) with those of aspirin itself. AZN was synthesized and interacted with a model transport protein, human serum albumin (HSA). Three‐dimensional fluorescence, ultraviolet–visible and circular dichroism (CD) spectra were used to characterize the interaction of AZN with HSA under physiological conditions. The interaction mechanism was explored using a fluorescence quenching method and thermodynamic calculation. The binding site and binding locality of AZN on HSA were demonstrated using a fluorescence probe technique and Förster non‐radiation energy transfer theory. Synchronous fluorescence and CD spectra were employed to reveal the effect of AZN on the native conformation of the protein. The HSA‐binding results for AZN were compared with those for aspirin under consistent experimental conditions, and indicated that aspirin acts as a guide in AZN when binding to Sudlow's site I, in subdomain IIA of the HSA molecule. Moreover, compared with aspirin, AZN showed greater observed binding constants with, but smaller changes in the α‐helicity of, HSA, which proved that AZN might be easier to transport and have less toxicity in vivo.     

Analysis of binding interaction between the natural apocarotenoid bixin and human serum albumin by circular dichroism and fluorescence spectroscopy

Bixin is an important, pharmacologically active dietary cis-carotenoid, but its interaction with potential macromolecular targets is completely unexplored. This work was aimed to study the binding of bixin to human serum albumin (HSA), the most abundant protein in blood plasma. Circular dichroism (CD) spectroscopy in combination with UV/VIS absorption spectroscopy and fluorescence quenching techniques were applied. Appearance of induced CD bands in the UV- and VIS-absorption spectral regions indicated the formation of non-covalent carotenoid-albumin complexes. Shape and spectral position of the extrinsic Cotton effects suggested the binding of a single bixin molecule to HSA in chiral conformation. Scatchard and non-linear regression analyses of CD titration data resulted in similar values for the association constant (Ka = 6.6 and 4.6x10(5) M(-1), resp.) and for the number of binding sites (n = 1). The binding interaction was independently confirmed by fluorescence-quenching experiment from which the binding parameters were also calculated. CD Displacement measurements performed with marker ligands established that the main drug binding sites of HSA are not involved in binding of bixin. Palmitic acid decreased the amplitude of the induced CD bands suggesting a common albumin binding site for bixin and long-chain fatty acids. The above data indicate that HSA plays a significant role in the plasma transportation of bixin and related dietary carboxylic acid carotenoids.     

Study of fluorescence interaction and conformational changes of bovine serum albumin with histamine H1‐receptor–drug epinastine hydrochloride by spectroscopic and time‐resolved fluorescence methods

The fluorescence, ultraviolet (UV) absorption, time resolved techniques, circular dichroism (CD), and infrared spectral methods were explored as tools to investigate the interaction between histamine H₁ drug, epinastine hydrochloride (EPN), and bovine serum albumin (BSA) under simulated physiological conditions. The experimental results showed that the quenching of the BSA by EPN was static quenching mechanism and also confirmed by lifetime measurements. The value of n close to unity indicated that one molecule of EPN was bound to protein molecule. The binding constants (K) at three different temperatures were calculated (7.1 × 10⁴, 5.5 × 10⁴, and 3.9 × 10⁴M⁻¹). Based on the thermodynamic parameters (ΔH⁰, ΔG⁰, and ΔS⁰), the nature of binding forces operating between drug and protein was proposed. The site of binding of EPN in the protein was proposed to be Sudlow's site I based on displacement experiments using site markers viz, warfarin, ibuprofen, and digitoxin. Based on the Förster's theory of non‐radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (EPN) was evaluated and found to be 4.48 nm. The UV–visible, synchronous fluorescence, CD, and three‐dimensional fluorescence spectral results revealed the changes in secondary structure of the protein upon its interaction with EPN. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 646–657, 2015.     

Circular dichroism and absorption spectroscopic data reveal binding of the natural cis-carotenoid bixin to human alpha1-acid glycoprotein

Using circular dichroism (CD) and electronic absorption spectroscopy techniques, interaction of the natural dietary cis-carotenoid bixin with an important human plasma protein in vitro was demonstrated for the first time. The induced CD spectra of bixin obtained under physiological conditions (pH 7.4, 37 degrees C) revealed its binding to the serum acute-phase reactant alpha(1)-acid glycoprotein (AGP), a member of the lipocalin protein family. Spectral features of the extrinsic Cotton effects of bixin suggested the inclusion of a single, chirally distorted ligand molecule into the asymmetric protein environment. Compared with the absorption spectra obtained in ethanol and benzene, the strong red shift of the main absorption peak of AGP-bound bixin indicated that the proposed binding site was rich in aromatic residues, and also suggested that hydrophobic interactions were involved in the binding. Using the data obtained from the CD titration experiments, the association constant (Ka=4.5x10(5)M-1) and stoichiometry of the binding (0.15) were calculated. The low value of the stoichiometry was attributed to the structural polymorphism of AGP. To the authors' knowledge, the current study represents the first human lipocalin protein for which carotenoid binding affinity has been explored in vitro with these techniques.     

Spectroscopic study on the interaction of bovine serum albumin with zinc(II) phthalocyanine

The interaction between the photosensitive antitumour drug, 2(3),9(10),16(17),23(24)‐tetra‐(((2‐aminoethylamino)methyl)phenoxy)phthalocyaninato‐zinc(II) (ZnPc) and bovine serum albumin (BSA) has been investigated using various spectroscopic methods. This work may provide some useful information for understanding the interaction mechanism of anticancer drug–albumin binding and gain insight into the biological activity and metabolism of the drug in blood. Based on analysis of the fluorescence spectra, ZnPc could quench the intrinsic fluorescence of BSA and the quenching mechanism was static by forming a ground state complex. Meanwhile, the Stern–Volmer quenching constant (K_SV), binding constant (K_b), number of binding sites (n) and thermodynamic parameters were obtained. Results showed that the interaction of ZnPc with BSA occurred spontaneously via hydrogen bond and van der Waal's force. According to Foster's non‐radioactive energy transfer theory, the energy transfer from BSA to ZnPc occurred with high possibility. Synchronous fluorescence and circular dichroism (CD) spectra also demonstrated that ZnPc induced the secondary structure of and conformation changes in BSA, especially α helix. Copyright © 2015 John Wiley & Sons, Ltd.     

Mechanistic and conformational studies on the interaction of sulfamethazine with human immunoglobulin G by molecular modeling and multi‐spectroscopic approach in vitro

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ ? HIgG complex. Fluorescence spectroscopy, ultraviolet–visible (UV–vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?19.12 kJ · mol^?1 and 20.22 J · mol^?1 · K^?1, respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ ? HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three‐dimensional fluorescence spectroscopy, UV–vis absorption spectroscopy and CD spectroscopy. Copyright © 2014 John Wiley & Sons, Ltd.     

Interaction of isofraxidin with human serum albumin

This study was designed to examine the interaction of isofraxidin with human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 3.3 x 10(-6) mol L(-1)-3.0x10(-5) mol L(-1) and HSA concentration at 1.5 x 10(-6) mol L(-1). Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy were used to determine the drug-binding mode, the binding constant and the protein structure changes in the presence of isofraxidin in aqueous solution. Spectroscopic evidence showed that the interaction results in one type of isofraxidin-HSA complex with binding constants of 4.1266 x 10(5) L mol(-1), 3.8612 x 10(5) L mol(-1), 3.5063 x 10(5) L mol(-1), 3.1241 x 10(5) L mol(-1) at 296 K, 303 K, 310 K, 318 K, respectively. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS) were calculated to be -10.08 kJ mol(-1) and 73.57 J mol(-1) K(-1) according to van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to HSA. The experiment results are nearly in accordance with the calculation results obtained by Silicon Graphics Ocatane2 workstation.