Investigation of the binding of Salvianolic acid B to human serum albumin and the effect of metal ions on the binding

The studies on the interaction between HSA and drugs have been an interesting research field in life science, chemistry and clinical medicine. There are also many metal ions present in blood plasma, thus the research about the effect of metal ions on the interaction between drugs and plasma proteins is crucial. In this study, the interaction of Salvianolic acid B (Sal B) with human serum albumin (HSA) was investigated by the steady-state, synchronous fluorescence and circular dichroism (CD) spectroscopies. The results showed that Sal B had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. Binding parameters calculated showed that Sal B was bound to HSA with the binding affinities of 10(5) L mol(-1). The thermodynamic parameters studies revealed that the binding was characterized by positive enthalpy and positive entropy changes, and hydrophobic interactions were the predominant intermolecular forces to stabilize the complex. The specific binding distance r (2.93 nm) between donor (HSA) and acceptor (Sal B) was obtained according to F?rster non-radiative resonance energy transfer theory. The synchronous fluorescence experiment revealed that Sal B cannot lead to the microenvironmental changes around the Tyr and Trp residues of HSA, and the binding site of Sal B on HSA is located in hydrophobic cavity of subdomain IIA. The CD spectroscopy indicated the secondary structure of HSA is not changed in the presence of Sal B. Furthermore, The effect of metal ions (e.g. Zn(2+), Cu(2+), Co(2+), Ni(2+), Fe(3+)) on the binding constant of Sal B-HSA complex was also discussed.     

Interaction Between Toddalolatone and Human Serum Albumin

A combination of fluorescence, UV–Vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopic and molecular modeling approaches was employed to investigate the interaction between toddalolactone (TDT) and human serum albumin (HSA) at physiological buffer conditions (pH 7.4). Fluorescence titration suggests that the mechanism of the fluorescence quenching of HSA is static, resulting from the formation of a TDT–HSA complex. Binding parameters calculated from the modified Stern–Volmer equation show that TDT binds to HSA with high affinity. Negative enthalpy change and positive entropy change values suggest that the binding process is primarily driven by hydrophobic interactions and hydrogen bonds. The binding of TDT to HSA results in an increase in the surface hydrophobicity of HSA. The binding distance between the Trp-214 residue (donor) and TDT (acceptor) was determined to be 4.18 nm based on the Förster theory of non-radioactive energy transfer. Displacement studies of site markers reveal that the binding site of TDT to HSA is located in the subdomain IIA (Sudlow’s site I). Furthermore, the molecular docking results corroborate and illustrate the specific binding mode and binding site. Analysis of UV–Vis absorption, CD and FT-IR spectra demonstrated that TDT induced a small alteration of the protein’s conformation.     

芘与人血清白蛋白和牛血清白蛋白结合位点微环境极性的差异

利用芘(Pyr)的微环境极性探针性质, 采用稳态荧光光谱、 荧光共振能量转移技术结合分子对接法, 对比分析了Pyr分别与人血清白蛋白(HSA)和牛血清白蛋白(BSA)作用机制的差异. 结果表明, HSA和BSA中Pyr的I₁/I₃平均值分别为1.36和0.92; Pyr与HSA和BSA的结合常数分别为1.86×10⁷和1.71×10⁵ L/mol; Pyr与HSA和BSA中色氨酸残基表观距离分别为2.37和2.34 nm. Pyr在HSA和BSA中不同的结合位点位于ⅠB子域和ⅠA子域, 其结合位点周围氨基酸残基的极性是影响Pyr I₁/I₃值的主要原因之一. 实验证实Pyr与HSA和BSA结合作用位点处的微环境极性存在差异.     

Protein binding of quinolonecarboxylic acids. II. Spectral changes on the interaction of cinoxacin, nalidixic acid and pipemidic acid with human and rat albumins

The interaction of cinoxacin (CINX), nalidixic acid (NA), and pipemidic acid (PPA) with human and rat serum albumins (HSA and RSA) was studied by UV difference absorption and circular dichroism (CD) spectroscopy. CINX and NA bound to the albumins and generated difference absorption and induced CD (ICD) spectra. The difference absorption spectral data explained reasonably our previous observations that CINX bound to HSA more weakly than NA, but to RSA as strongly as NA. We used a quantity delta epsilon/epsilon, designated as relative molar difference absorbance, at positions corresponding to the longest wavelength peaks in the difference spectra. The quantity was found to correlate linearly with percent bound to both HSA and RSA, but with different slopes, from which the binding site for CINX and NA in RSA was supposed to provide a much more nonpolar environment than that in HSA. The magnitude of ICD bands observed at 371 nm for CINX and at 342-348 nm for NA corresponded to the binding degrees of these drugs to both albumins. Anisotropy factors for the ICD bands at 350-271 nm for CINX and 320-348 nm for NA were approximately similar between HSA and RSA, suggesting a similar ability to generate the ICD spectra in these wavelength regions upon binding to the albumins. Spectral results for PPA in albumin solutions showed little or no binding of this drug to HSA and RSA. PPA existed as a betaine form in neutral solution and its positively charged group acted as an unfavorable factor for binding to both albumins.     

Studies on the interaction of cinnamic acid with bovine serum albumin

The interaction between cinnamic acid and bovine serum albumin (BSA) have been studied at three temperatures, 296, 303 and 310 K. Fluorescence quenching spectra in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to investigate the drug-binding mode, the binding constant and the protein structure changes in the presence of cinnamic acid in aqueous solution at pH 7.40. The fluorescence quenching constant K(q), K(sv) and the binding constant K were calculated according to Stern-Volmer equation based on the quenching of the fluorescence of BSA in the presence of cinnamic acid. The thermodynamic parameters, the enthalpy (DeltaH) and the entropy change (DeltaS) were estimated to be -16.457 kJ mol(-1) and 38.028 J mol(-1) K(-1) according to the van't Hoff equation. The displacement experiment shows that cinnamic acid can bind to the subdomain IIA (corresponding to Sudlow's drug binding site I). The distance between the tryptophan residues in BSA and cinnamic acid bound to site I was estimated to be 1.63 nm using F?ster's equation on the basis of fluorescence energy transfer. The decreased binding constant in the presence of common ions indicates that common ions have effect on drug-BSA system.     

Investigation on the interaction between a heterocyclic aminal derivative, SBDC, and human serum albumin

The interaction between a novel promising drug (spiro[(2R,3R,4S)-4-benzyloxy-2,3-isopropylidene-dioxy-1-oxa-cyclopentane-5,5′-(2-benzoylmethylene-1,3-diaza-cyclohexane)] (SBDC)) and human serum albumin (HSA) under physiological conditions has been investigated by using fluorescence, absorption, and circular dichroism (CD) spectroscopic techniques in combination with protein–ligand docking study. It was observed that SBDC has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The association constants of SBDC with HSA were determined at different temperatures based on fluorescence quenching results. The negative ΔH and positive ΔS values in case of SBDC–HSA complex showed that apart from an initial hydrophobic association, both van der Waals interactions and hydrogen bonding play a vital role in the binding of SBDC to HSA. The quantitative analysis data of CD spectra showed that the binding of SBDC to HSA induced conformational changes in HSA and the α-helix of 52.1% in free HSA increased to 55.7% in HSA–SBDC complex. The distance between donor (HSA) and acceptor (SBDC) was obtained according to the Förster's theory of non-radiation energy transfer. Data obtained by spectroscopic techniques and protein–ligand docking study suggested that SBDC binds to residues located in subdomain IIA of HSA.     

Chiral complexation and aggregation of bilirubin with serum albumin at a liquid/liquid interface

The chiral complexation of bilirubin (BR) with bovine and human serum albumin (BSA and HSA), and the aggregation of the complexes at the heptane+chloroform(5:1)/water interface were studied via UV/Vis absorption and circular dichroism (CD) measurements in combination with the centrifugal liquid membrane (CLM) method. The interfacial adsorptivities of BR, BSA and their complexes were also studied by performing interfacial tension measurements at the interface. The changes in the absorbances and the induced CD amplitudes of the interfacial BR-BSA complex provided insights into the mechanism of the conformational enantioselective complexation at the interface, and indicated that the chiral conversion induced by the complexation with BSA was from the P(+) form to the M(-) form of BR. The broadening of the 450 nm band and the appearance of a new shoulder at 474 nm further supported the formation of aggregates of the complexes at the interface. The dependence of the CD amplitude on the molar ratio of BSA to BR revealed that the composition of the complex was 1:1 BSA:BR. The probable interfacial reaction scheme was proposed, and the affinity constant of BR-BSA at the interface was found to be 4.67 x 10(8) M(-2). The interfacial complexation and aggregation of BR and HSA were weaker than those of the BR-BSA complex due to the different BR binding positions adopted for BSA and HSA and the binding effect of chloroform.     

Investigation of the behavior of HSA upon binding to amlodipine and propranolol: Spectroscopic and molecular modeling approaches

The interaction between human serum albumin (HSA) and two drugs - amlodipine and propranolol - was investigated using fluorescence, UV absorption and circular dichroism (CD) spectroscopy. In addition, the binding site was established by applying molecular modeling technique. Fluorescence data suggest that amlodipine will quench the intrinsic fluorescence of HSA; whereas propranolol enhances the fluorescence of HSA. The binding constants for the interaction of amlodipine and propranolol with HSA were found to be 3.63×10(5)M(-1) and 2.29×10(4)M(-1), respectively. The percentage of secondary structure feature of each one of the HSA-bound drugs, i.e. the α-helix content, was estimated empirically by circular dichroism. The results indicated that amlodipine causes an increase, and that propranolol leads to a decrease in α-helix content of HSA. The spectroscopic analysis indicates that the binding mechanisms of the two drugs are different from each other. The data obtained by the molecular modeling study indicated that these drugs bind, with different affinity, to different sites located in subdomain IIA and IIIA.     

光谱法研究氨基己酸与牛血清白蛋白的相互作用

利用荧光光谱、紫外-可见吸收光谱及圆二色（CD）光谱研究了模拟生理条件下的氨基己酸(ACA)与牛血清白蛋白(BSA)的相互作用。 实验结果分析表明,氨基己酸对BSA的内源性荧光具有猝灭作用,属于动态猝灭过程。 计算了2种温度下ACA-BSA体系的结合常数、结合位点数及反应的热力学参数ΔG、ΔH和ΔS分别约为-21.00 kJ/mol、-0.64 kJ/mol和-72.00 kJ/(mol·K),由此推出了二者主要通过氢键和范德华力形成摩尔比为1∶1的复合物。 依据Forster非辐射能量转移理论求得二者之间的结合距离为2.3 nm。 位点取代实验指出氨基己酸主要结合在位点Site I。 CD光谱表明,氨基己酸诱导了BSA分子二级结构微变。     

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.     

Binding of Dioxopromethazine Hydrochloride with Human Serum Albumin and Its Effect on the Conformation of the Protein

The interaction between dioxopromethazine hydrochloride (DPZ) and human serum albumin (HSA), in vitro, was investigated by means of fluorescence, absorption and circular dichroism (CD) spectroscopy. The results obtained from analysis of the fluorescence spectra indicate that DPZ has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. The HSA?CDPZ binding distance was determined to be less than 7?nm, suggesting that energy transfer from HSA to DPZ may occur. The thermodynamic parameters of the interaction between DPZ and HSA were measured according to the van??t Hoff equation. The negative enthalpy change (??H ⁰) and positive entropy change (??S ⁰) values indicate that hydrophobic and electrostatic interactions play major roles in the binding between DPZ and HSA. The binding process was a favorable process in which the Gibbs energy change (??G ⁰) is negative. The results of displacement experiments suggested that DPZ is located in site I of HSA within subdomain IIA. In addition, the results of absorption, synchronous fluorescence and CD spectra show that binding of DPZ with HSA can induce conformational changes in HSA.     

Synthesis and interaction studies of benzimidazole derivative with human serum albumin

A benzimidazole derivative, 1-(2-picolyl)-3-(2-picolyl) benzimidazole iodide (PPB), was synthesized. Fourier transform infrared spectroscopy (FT-IR), UV–visible, three-dimensional (3D) fluorescence, synchronous fluorescence (SF) and fluorescence spectroscopic methods were used to determine the PPB binding mode and the effects of PPB on protein stability and secondary structure. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of PPB to human serum albumin (HSA). The binding constants between PPB and HSA were obtained according to Scatchard equation. The number of binding sites, the binding constants and the thermodynamic parameters were measured. The results showed a spontaneous binding of PPB to HSA through hydrogen bonds and van der Waals forces. In addition, the distance between PPB and the Trp 214 was estimated via employing the Förster's non-radiative energy transfer theory, and was found to be 3.49 nm, which indicated that PPB can bind to HSA with high probability. Site marker competitive experiments indicated that the binding of PPB to HSA primarily took place in subdomain IIA.     

In vitro study on the binding of herbicide glyphosate to human serum albumin by optical spectroscopy and molecular modeling

In this study, the interaction mechanism of herbicide glyphosate and human serum albumin (HSA) has been characterized by fluorescence, UV, Fourier transform infrared (FT-IR), Circular dichroism (CD) spectroscopic and molecular modeling methods. The structural characteristics of glyphosate and HSA were probed, and affinity constants were determined under different temperatures. The enthalpy change (DeltaH degrees ) and the entropy changes (DeltaS degrees ) were calculated to be -21.78kJmol(-1) and 6.38Jmol(-1)K(-1) according to the Van't Hoff equation. These results indicated that glyphosate binds to HSA mainly by hydrogen bond and hydrophobic interaction can also not be excluded, which is in good agreement with the results from modeling experiment. The average binding distance, r, between the donor (HSA) and the acceptor (glyphosate) was evaluated and found to be 2.89nm according to the F?rster's theory of non-radiation energy transfer. The alterations of protein secondary structure in the presence of glyphosate were confirmed by the evidences from UV, FT-IR and CD spectroscopes.     

Structure and properties of licochalcone A-human serum albumin complexes in solution: a spectroscopic, photophysical and computational approach to understand drug-protein interaction

In the present contribution we address the study of the interaction of a flavonoid-derivative licochalcone A (LA) with human serum albumin (HSA). The application of circular dichroism, UV-Vis absorption, fluorescence and laser flash photolysis combined with molecular mechanics, molecular dynamics and quantum mechanical calculations of rotational strength afforded a clear picture of the modes of association of the LA neutral molecule to HSA, evidencing specific interactions with protein amino acids and their photophysical consequences. The drug is primarily associated in subdomain IIA where a strong interaction with Trp214 is established. At least two different positions of LA with respect to tryptophan are possible, one with the phenolic ring of the drug facing the aromatic ring of Trp214 and the other with the methoxyphenolic ring of LA in proximity to Trp214. In both cases LA is at ca. 4 angstroms from Trp214. This vicinity does not affect much the S1 singlet state deactivation of the bound drug, which exhibits a slightly higher fluorescence quantum yield and fluorescence lifetime on the order of that of the free molecule. The LA triplet lifetime appears to be somewhat shortened in this site. The secondary binding site is in subdomain IIIA. Here, the carbonyl group of LA experiences a strong H-bond with the OH-phenolic substituent of Tyr411. This interaction reduces substantially the LA molecular degrees of freedom, thereby determining a decrease of both radiative and nonradiative rate constants for decay of the singlet. The overall rigidity of the structure causes a lengthening of the triplet lifetime.     

光谱法与分子模拟对左旋紫草素和人血白蛋白键合作用的研究

采用荧光光谱法、紫外吸收光谱法和圆二色性光谱(CD)研究了模拟生理条件下左旋紫草素和人血清白蛋白(HSA)的相互作用,计算了反应的结合常数、结合位点数和热力学参数,并探讨了左旋紫草素对人血清白蛋白二级结构的影响.在温度为292、303、310和318 K时,根据Scatchard方程测得左旋紫草素和HSA的结合常数分别为3.118×10~6、0.249×10~6、0.112×10~6 和0.102×10~6 L·mol~(-1),结合位点数分别为1.308、1.094、1.026和1.018;焓变(ΔH)和熵变(ΔS)分别为-104.82 kJ·mol~(-1)、-238.18 J·mol~(-1)·K~(-1),左旋紫草素在人血清白蛋白上的结合位置与色氨酸残基间的距离为2.66 nm.分子模型研究表明,左旋紫草素与HSA在亚结构域ⅡA结合,二者间的作用力主要为疏水和氢键作用力.CD结果表明,左旋紫草素与HSA的键合使HSA中α-螺旋结构含量从55.80%降到52.31%.     

Synthesis,structural determination and HSA interaction studies of a new water-soluble Cu(II) complex derived from 1,10-phenanthroline and ranitidine drug

A new water-soluble Cu(II) complex containing ranitidine drug and 1,10-phenanthroline was synthesized and characterized by elemental analysis, molar conductivity, spectroscopic and computational methods. In vitro human serum albumin (HSA)-interaction studies of Cu(II) complex were performed by employing fluorescence spectroscopy in combination with UV–vis absorption and circular dichroism (CD) spectroscopies. The results of fluorescence titration showed that Cu(II) complex strongly quenched the intrinsic fluorescence of HSA through a static quenching mechanism with an intrinsic binding constant (6.05 × 10⁴ M^?1) at 286 K. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated and suggested that the hydrophobic and hydrogen bonding interactions play major roles in Cu(II) complex-HSA association. The displacement experiments using warfarin and ibuprofen as site I and II probes proved that the Cu(II) complex could bind to site I (subdomain IIA) of HSA. Finally, CD spectra indicated that the interaction of the Cu(II) complex with HSA leads to an increase in the α-helical content. The main result of this study was the finding that the binding affinity of the Cu(II) complex to HSA is three orders of magnitude stronger than that of ranitidine drug.     

Spectroscopic studies and life time measurements of binding of a bioactive compound to bovine serum albumin and the effects of common ions and other drugs on binding

The mechanism of binding of anti-inflammatory drug, nimesulide (NIM) with bovine serum albumin (BSA) was investigated by fluorescence, absorption, circular dichroism (CD) and lifetime measurements under simulative physiological conditions. The analysis of fluorescence data indicated the presence of both dynamic and static quenching mechanism in the binding. Various binding parameters have been evaluated. The CD spectral data revealed the decrease in alpha-helical content of BSA from 70.9% (in free BSA) to 42.03% (in bound form) thereby indicating the conformational change in BSA upon binding. The binding of NIM to BSA was also confirmed by absorption spectra. Based on the F?rster's theory of non-radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (NIM) was found to be 2.17 nm. The association constants of NIM-BSA decreased in presence of the common ions and other drugs thereby indicating the availability of higher concentration of free drug (NIM) in plasma.     

Binding of the bioactive component daphnetin to human serum albumin demonstrated using tryptophan fluorescence quenching

Daphnetin (7,8-dihydroxycoumarin), one of the major bioactive components isolated from Daphne koreane Nakai, has been used in traditional Chinese medicine for the treatment of coagulation disorders. It is also a chelator, an antioxidant and a protein kinase inhibitor. In this paper, a combination of intrinsic fluorescence, Fourier transform infrared (FT-IR) spectroscopy and circular dichroic (CD) spectroscopy has been used to characterize the binding between daphnetin and human serum albumin (HSA) under physiological conditions with drug concentrations of 6.7 x 10(-6) - 2.3 x 10(-5) mol x L(-1), and a HSA concentration of 1.5 x 10(-6) mol x L(-1). Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching did change significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (DeltaH) and the entropy change (DeltaS) were calculated to be -12.45 kJ x mol(-1)and 52.48 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic and electrostatic interactions played the main role in the binding of daphnetin to HSA, in accordance with the results of calculations performed on a Silicon Graphics Ocatane2 workstation. In addition, the binding distance between daphnetin and HSA was obtained (4.02 nm) based on the Forster energy transfer theory.     

Transient absorption spectroscopy for determining multiple site occupancy in drug-protein conjugates. A comparison between human and bovine serum albumins using flurbiprofen methyl ester as a probe

Laser flash photolysis (LFP) has been used to determine the degree of binding of (S)- or (R)-flurbiprofen methyl ester (FBPMe) to human and bovine serum albumins (HSA and BSA, respectively). Regression analysis of the triplet decay of the drug (lambda = 360 nm) in the presence of the proteins led to a satisfactory fitting when considering a set of three lifetimes; the corresponding Afree, AI and AII preexponential coefficients can be correlated with the presence of FBPMe in the bulk solution and within the two known binding sites. The most remarkable differences between HSA and BSA were found under nonsaturating conditions; thus, when the [FBPMe]/[SA] ratio was 1:1, all the drug was bound to HSA, whereas 20-30% of it remained free in the bulk solution in the presence of BSA. The LFP approach was also applicable to the study of more complex FBPMe/HSA/BSA mixtures; the obtained results were in good agreement with the previous findings in FBPMe/HSA and FBPMe/BSA systems. This suggests the possibility of making use of the transient triplet-triplet absorption for investigating the distribution of a drug between several compartments in different host biomolecules.