Interaction Study between ESIPT Fluorescent Lipophile-Based Benzazoles and BSA

In this study, the interactions of ESIPT fluorescent lipophile-based benzazoles with bovine serum albumin (BSA) were studied and their binding affinity was evaluated. In phosphate-buffered saline (PBS) solution these compounds produce absorption maxima in the UV region and a main fluorescence emission with a large Stokes shift in the blue–green regions due to a proton transfer process in the excited state. The interactions of the benzazoles with BSA were studied using UV-Vis absorption and steady-state fluorescence spectroscopy. The observed spectral quenching of BSA indicates that these compounds could bind to BSA through a strong binding affinity afforded by a static quenching mechanism (K_q~10¹² L·mol⁻¹·s⁻¹). The docking simulations indicate that compounds 13 and 16 bind closely to Trp134 in domain I, adopting similar binding poses and interactions. On the other hand, compounds 12, 14, 15, and 17 were bound between domains I and III and did not directly interact with Trp134.     

Spectroscopic Studies on the Binding of Kaempferol‐3,7‐α‐L‐rhamnopyranoside to Bovine Serum Albumin

The binding of kaempferol‐3,7‐α‐L‐rhamnopyranoside (KRR) with bovine serum albumin (BSA) was investigated by different spectroscopic methods under simulative physiological conditions. Analysis of ?uorescence quenching data of BSA by KRR at different temperatures using Stern‐Volmer methods revealed the formation of a ground state KRR‐BSA complex with moderate binding constant of the order 10⁴ L·mol^?1. The existence of some metal ions could weaken the binding of KRR on BSA. The changes in the van't Hoff enthalpy (ΔH⁰) and entropy (ΔS⁰) of the interaction were estimated to be ?26.53 kJ·mol^?1 and 3.33 J·mol^?1·K^?1 and both hydrophobic and electrostatic forces contributed to stabilizing the BSA‐KRR complex. According to the F?ster theory of non‐radiation energy transfer, the distance r between the donor (BSA) and the acceptor (KRR) was obtained (r=2.83 nm). Site marker competitive experiments showed that KRR could bind to Site I of BSA. In addition, synchronous fluorescence, UV‐Vis absorption and circular dichroism (CD) results indicated that the KRR binding could cause conformational changes of BSA.     

Interaction between Curcumin and β-Casein: Multi-Spectroscopic and Molecular Dynamics Simulation Methods

Effect of temperature and pH on the interaction of curcumin with β-casein was explored by fluorescence spectroscopy, ultraviolet-visible spectroscopy and molecular dynamics simulation. The spectroscopic results showed that curcumin could bind to β-casein to form a complex which was driven mainly by electrostatic interaction. The intrinsic fluorescence of β-casein was quenched by curcumin through static quenching mechanism. The binding constants of curcumin to β-casein were 6.48 × 10⁴ L/mol (298 K), 6.17 × 10⁴ L/mol (305 K) and 5.73 × 10⁴ L/mol (312 K) at pH 2.0, which was greater than that (3.98 × 10⁴ L/mol at 298 K, 3.90 × 10⁴ L/mol at 305 K and 3.41 × 10⁴ L/mol at 312 K) at pH 7.4. Molecular docking study showed that binding energy of β-casein-curcumin complex at pH 2.0 (−7.53 kcal/mol) was lower than that at pH 7.4 (−7.01 kcal/mol). The molecular dynamics simulation study showed that the binding energy (−131.07 kJ/mol) of β-casein-curcumin complex was relatively low at pH 2.0 and 298 K. α-Helix content in β-casein was decreased and random coil content was increased in the presence of curcumin. These results can promote a deep understanding of interaction between curcumin and β-casein and provide a reference for improving the bioavailability of curcumin.     

Understanding the Impact of Key Wine Components on the Use of a Non-Swelling Ion-Exchange Resin for Wine Protein Fining Treatment

The impact of key classes of compounds found in wine on protein removal by the ion-exchange resin, Macro-Prep^® High S, was examined by adsorption isotherm experiments. A model wine system, which contained a prototypical protein Bovine Serum Albumin (BSA), was used. We systematically changed concentrations of individual chemical components to generate and compare adsorption isotherm plots and to quantify adsorption affinity or capacity parameters of Macro-Prep^® High S ion-exchange resin. The pH (hydronium ion concentration), ethanol concentration, and prototypical phenolics and polysaccharide compounds are known to impact interactions with proteins and thus could alter the adsorption affinity and capacity of Macro-Prep^® High S ion-exchange resin. At low equilibrium protein concentrations (< ~0.3 (g BSA)/L) and at high equilibrium protein concentrations in model wines at various pH, the adsorption behavior followed the Langmuir isotherm, most likely due to the resin acting as a monolayer adsorbent. The resulting range of BSA capacity was between 0.15–0.18 (g BSA)/(g Macro-Prep^® High S resin). With the addition of ethanol, catechin, caffeic acid, and polysaccharides, the protein adsorption behavior was observed to differ at higher equilibrium protein concentrations (> ~0.3 (g BSA)/L), likely as a result of Macro-Prep^® acting as an unrestricted multilayer adsorbent at these conditions. These data can be used to inform the design and scale-up of ion-exchange columns for removing proteins from wines.     

Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC)

This work aims at understanding the attachment mechanisms and stability of proteins on a chromatography medium to develop more efficient functionalization methodologies, which can be exploited in affinity chromatography. In particular, the study was focused on the understanding of the attachment mechanisms of bovine serum albumin (BSA), used as a ligand model, and protein G on novel amine-modified alumina monoliths as a stationary phase. Protein G was used to develop a column for antibody purification. The results showed that, at lower protein concentrations (i.e., 0.5 to 1.0 mg·mL⁻¹), protein attachment follows a 1st-order kinetics compatible with the presence of covalent binding between the monolith and the protein. At higher protein concentrations (i.e., up to 10 mg·mL⁻¹), the data preferably fit a 2nd-order kinetics. Such a change reflects a different mechanism in the protein attachment which, at higher concentrations, seems to be governed by physical adsorption resulting in a multilayered protein formation, due to the presence of ligand aggregates. The threshold condition for the prevalence of physical adsorption of BSA was found at a concentration higher than 1.0 mg·mL⁻¹. Based on this result, protein concentrations of 0.7 and 1.0 mg·mL⁻¹ were used for the functionalization of monoliths with protein G, allowing a maximum attachment of 1.43 mg of protein G/g of monolith. This column was then used for IgG binding–elution experiments, which resulted in an antibody attachment of 73.5% and, subsequently, elution of 86%, in acidic conditions. This proved the potential of the amine-functionalized monoliths for application in affinity chromatography.     

Interaction of Carrier Protein with Potential Metallic Drug Candidate N-Glycoside ‘GATPT’: Validation by Multi-Spectroscopic and Molecular Docking Approaches

Lysozyme is often used as a model protein to study interaction with drug molecules and to understand biological processes which help in illuminating the therapeutic effectiveness of the drug. In the present work, in vitro interaction studies of 1-{(2-hydroxyethyl)amino}-2-amino-1,2-dideoxy-d-glucose triphenyl tin (IV) (GATPT) complex with lysozyme were carried out by employing various biophysical methods such as absorption, fluorescence, and circular dichroism (CD) spectroscopies. The experimental results revealed efficient binding affinity of GATPT with lysozyme with intrinsic binding (K_b) and binding constant (K) values in the order of 10⁵ M⁻¹. The number of binding sites and thermodynamic parameters ΔG, ΔH, and ΔS at four different temperatures were also calculated and the interaction of GATPT with lysozyme was found to be enthalpy and entropy driven. The CD spectra revealed alterations in the population of α–helical content within the secondary structure of lysozyme in presence of GATPT complex. The morphological analysis of the complex with lysozyme and lysozyme-DNA condensates was carried out by employing confocal and SEM studies. Furthermore, the molecular docking studies confirmed the interaction of GATPT within the larger hydrophobic pocket of the lysozyme via several non-covalent interactions.     

Screening of Potential Thrombin and Factor Xa Inhibitors from the Danshen–Chuanxiong Herbal Pair through a Spectrum–Effect Relationship Analysis

In this study; a spectrum–effect relationship analysis combined with a high-performance liquid chromatography–mass spectrometry (LC–MS) analysis was established to screen and identify active components that can inhibit thrombin and factor Xa (THR and FXa) in Salviae Miltiorrhizae Radix et Rhizoma–Chuanxiong Rhizoma (Danshen–Chuanxiong) herbal pair. Ten potential active compounds were predicted through a canonical correlation analysis (CCA), and eight of them were tentatively identified through an LC–MS analysis. Furthermore; the enzyme inhibitory activity of six available compounds; chlorogenic acid; Z-ligustilide; caffeic acid; ferulic acid; tanshinone I and tanshinone IIA; were tested to verify the feasibility of the method. Among them; chlorogenic acid was validated to possess a good THR inhibitory activity with IC₅₀ of 185.08 µM. Tanshinone I and tanshinone IIA are potential FXa inhibitors with IC₅₀ of 112.59 µM and 138.19 µM; respectively. Meanwhile; molecular docking results show that tanshinone I and tanshinone IIA; which both have binding energies of less than −7.0 kcal·mol⁻¹; can interact with FXa by forming H-bonds with residues of SER214; GLY219 and GLN192. In short; the THR and FXa inhibitors in the Danshen–Chuanxiong herbal pair have been successfully characterized through a spectrum–effect relationship analysis and an LC–MS analysis.     

Voltammetric Studies on Chromotrope 2B‐Protein Interaction and Its Analytical Application

In this paper the interaction of chromotrope 2B (Ch2B) with proteins was studied by the electrochemical method. Ch2B is an azo dye and shows irreversible electrochemical responses on the mercury electrode in a pH 3.0 Britton‐Robinson (B‐R) buffer solution. After the addition of human serum albumin (HSA) into the Ch2B solution, an interaction took place, and a supramolecular complex was formed in the mixed solution. The electrochemical parameters of the Ch2B‐HSA interaction system were calculated and compared. The results showed that in the absence and presence of HSA in Ch2B solution, the electrochemical parameters such as the formal potential E⁰, the electrode reaction standard rate constant k_s, etc. showed no significant changes, which indicated that an electro‐inactive supramolecular biocomplex was formed. The free concentration of Ch2B in reaction solution was decreased, and this resulted in the decrease of the peak current. The binding constant and the binding ratio were calculated as 7.85 × 10⁹ and 1:2, respectively, and the interaction mechanism was discussed. Based on the decrease of the peak current, this new electrochemical method was proposed for the determination of HSA in the concentration range of 2.0?25.0 mg/L with the linear regression equation as ΔIp′ (nA) = 50.56C (mg/L) — 6.72 (γ = 0.995). This method was further used to determine other different kinds of proteins, such as bovine serum albumin (BSA), oval albumin, etc‥ The new method was successfully applied to detect the content of albumin in healthy human serum samples with the results in good agreement with the traditional Coomassie Brilliant Blue G‐250 spectrophotometric method.     

Binding and Sensing Properties of a Hybrid Naphthalimide–Pyrene Aza-Cyclophane towards Nucleotides in an Aqueous Solution

Selective recognition of nucleotides with synthetic receptors is an emerging direction to solve a series of nucleic acid-related challenges in biochemistry. Towards this goal, a new aza-cyclophane with two different dyes, naphthalimide and pyrene, connected through a triamine linker has been synthesized and studied for the ability to bind and detect nucleoside triphosphates in an aqueous solution. The receptor shows Foerster resonance energy transfer (FRET) in fluorescence spectra upon excitation in DMSO, which is diminished dramatically in the presence of water. According to binding studies, the receptor has a preference to bind ATP (adenosine triphosphate) and CTP (cytidine triphosphate) with a “turn-on” fluorescence response. Two separate emission bands of dyes allow one to detect nucleotides in a ratiometric manner in a broad concentration range of 10⁻⁵–10⁻³ M. Spectroscopic measurements and quantum chemical calculations suggest the formation of receptor–nucleotide complexes, which are stabilized by dispersion interactions between a nucleobase and dyes, while hydrogen bonding interactions of nucleobases with the amine linkers are responsible for selectivity.     

Strength of the [Z–I···Hal]− and [Z–Hal···I]− Halogen Bonds: Electron Density Properties and Halogen Bond Length as Estimators of Interaction Energy

Bond energy is the main characteristic of chemical bonds in general and of non-covalent interactions in particular. Simple methods of express estimates of the interaction energy, E_int, using relationships between E_int and a property which is easily accessible from experiment is of great importance for the characterization of non-covalent interactions. In this work, practically important relationships between E_int and electron density, its Laplacian, curvature, potential, kinetic, and total energy densities at the bond critical point as well as bond length were derived for the structures of the [Z–I···Hal]⁻ and [Z–Hal···I]⁻ types bearing halogen bonds and involving iodine as interacting atom(s) (totally 412 structures). The mean absolute deviations for the correlations found were 2.06–4.76 kcal/mol.     

Binding Studies of a Schiff Base Compound Containing a 1,2,4-Triazole Ring with Bovine Serum Albumin Using Spectroscopic Methods

The binding interaction of a Schiff base compound containing a 1,2,4‐triazole ring [4‐(4‐chlorobenzyl‐ideneamino)‐5‐methyl‐1,2,4‐triazole‐3‐thiol, CTT] with bovine serum albumin (BSA) was studied by spectroscopy methods including fluorescence and circular dichroism spectrum under simulative physiological conditions. Fluorescence investigation revealed that the fluorescence quenching of BSA was induced by the formation of a relative stable CTT‐BSA complex. The corresponding binding constants (K_a) between CTT and BSA at three different temperatures were calculated according to the modified Stern‐Volmer equation. The enthalpy change (ΔH^⊖) and entropy change (ΔS^⊖) were calculated to be −15.78 kJ·mol⁻¹ and 49.23 J·mol⁻¹·K⁻¹, respectively, which suggested that hydrophobic forces and hydrogen bond played major roles in stabilizing the CTT‐BSA complex. Site marker competitive experiments indicated that the binding of CTT to BSA primarily took place in sub‐domain IIIA (site II) of BSA. The binding distance (r) between CTT and the tryptophan residue of BSA was obtained to be 4.3 nm based on F?rster theory of non‐radioactive energy transfer. The conformational investigation revealed that the presence of CTT decreased the α‐helix content of BSA (from 58.62% to 54.66%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro‐environmental and conformational changes of BSA molecules.     

Study of the conjugation reaction between bovine serum albumin and gentamicin with Ponceau S as fluorescence probe

Abstract  

Ponceau S (PS) can quench the fluorescence of bovine serum albumin (BSA) in aqueous solution of pH 7.40. The static fluorescence-quenching process between BSA and PS was confirmed and the binding constant, the number of binding sites, and thermodynamic data for the interaction between BSA and PS were obtained. The results showed that the number of binding sites was 1 and that electrostatic attraction was important in the binding of BSA to PS. On the basis of the theory of F?rster resonance energy transfer, the binding distance (r < 7 nm) between PS and BSA was obtained. Site marker competitive experiments indicated that binding of PS to BSA primarily occurred in sub-domain IIA (site I). There was no obvious fluorescence intensity change on combining BSA and gentamicin (GM), so the conjugation reaction between BSA and GM cannot be studied by spectroscopy. It was observed that when GM was added to the BSA–PS system, the relative fluorescence intensity of the system recovered gradually with increasing concentration of GM, which showed there was a conjugation reaction between GM and BSA and that binding of GM to BSA primarily occurred in sub-domain IIA (site I).     

Spectrophotofluorimetric Study of the Interaction between Trazodone Hydrochloride and Bovine Serum Albumin

Abstract

The binding of trazodone hydrochloride (TZH), an antidepressant drug, to bovine serum albumin (BSA) has been investigated by fluorescence spectroscopic analysis. The fluorescence emission of BSA (λ_em=350 nm) was quenched by TZH while that of this ligand was enhanced (λ_em=443 nm). The spectral behavior was consistent with the static quenching mechanism, and the apparent binding constant, K _a (1.05×10⁴ l mol^?1) as well as binding site number, n (～1), were estimated. Thermodynamic parameters obtained from the measured data at different temperatures showed that the binding of TZH to BSA involved predominantly hydrophobic interactions as well as smaller contributions from electrostatic forces. Phenylbutazone and ibuprofen were utilized as competitive markers for sites I and II, respectively, in the interaction of TZH with BSA. This competitive displacement procedure indicated that the likely binding was site I, i.e., subdomain IIA, and this was supported by the observation that up to 50% of this site marker, phenylbutazone, could be exchanged with TZH whilst only a few percent of ibuprofen were so affected.     

Multispectroscopic Studies on the Interaction of a Platinum(II) Complex Containing l-Histidine and 1,10-Phenanthroline Ligands with Bovine Serum Albumin

The mechanism of the interaction between bovine serum albumin (BSA) and [Pt(phen) (histidine)]⁺ complex was studied employing ultraviolet (UV) absorption, circular dichroism (CD), FT-IR, differential pulse voltammetry (DPV), and fluorescence spectral methods. Fluorescence data showed that the intrinsic fluorescence of BSA was strongly quenched by Pt(II) complex in terms of an untypical static quenching process. The corresponding number of binding sites (n) and binding constant (K _b) of BSA and complex at 283, 298, and 310 K were calculated to be 0.61?×?10⁶, 19?×?10⁶, and 42?×?10⁶ M^?1, respectively. The results showed that the increasing temperature improves the stability of the complex–BSA system, which results in a higher binding constant and the number of binding sites of the complex–BSA system. The positive ΔH and positive ΔS indicated that hydrophobic forces might play a major role in the binding between complex and BSA. Based on Forster’s theory of non-radiation energy transfer, the binding distance (r) between the donor (BSA) and acceptor (Pt(II) complex) was evaluated. The results of CD, UV–vis, DPV, and FT-IR spectroscopy showed that the binding of Pt(II) complex to BSA induced conformational changes in BSA     

Tween-20与牛血清白蛋白的相互作用

通过表面张力和等温滴定量热方法，研究了非离子表面活性剂Tween-20与牛血清白蛋白(BSA)的相互作用. 结果表明，BSA与Tween-20的相互作用较弱，当Tween-20浓度增大时，能将吸附于表面上的BSA分子取代. BSA/Tween-20混合体系的性质随温度的变化趋势与单一非离子型表面活性剂相似. BSA浓度在3.7×10^-5～11.1×10^-5 kg•L^-1范围内，Tween-20的cmc不随BSA浓度的变化而变化. 当Tween-20的含量一定时，Tween-20与BSA相互作用过程的热效应也与BSA 的浓度无关. BSA/Tween-20混合体系的热力学参数表明， BSA的存在使Tween-20的cmc减小，体系的熵变增大.     

羟丙基-β-环糊精对1-羟基芘与牛血清白蛋白相互作用的影响

在模拟生理条件下, 利用荧光光谱、 圆二色光谱和紫外-可见吸收光谱探究了羟丙基-β-环糊精(HPCD)对1-羟基芘(1-OHPyr)与牛血清白蛋白(BSA)相互作用的影响及其相关机制. 结果表明, 291 K下HPCD可使1-OHPyr与BSA的结合常数降低为1.45×10⁵ L/mol; 导致1-OHPyr-BSA体系中BSA的α-螺旋含量恢复, 色氨酸(TRP)残基周围微环境的极性变化减弱; 致使1-OHPyr及BSA的荧光寿命均延长, 且二者的结合距离增大. 初步研究结果表明, HPCD与1-OHPyr的包络作用是HPCD影响BSA与1-OHPyr结合的主要原因.     

Development and Validation of an HPLC-UV Method for the Quantification of 4′-Hydroxydiclofenac Using Salicylic Acid: Future Applications for Measurement of In Vitro Drug–Drug Interaction in Rat Liver Microsomes

Salicylic acid is a key compound in nonsteroidal anti-inflammatory drugs that has been recently used for preventing the risk of hospitalization and death among COVID-19 patients and in preventing colorectal cancer (CRC) by suppressing two key proteins. Understanding drug–drug interaction pathways prevent the occurrence of adverse drug reactions in clinical trials. Drug–drug interactions can result in the variation of the pharmacodynamics and pharmacokinetic of the drug. Inhibition of the Cytochrome P450 enzyme activity leads to the withdrawal of the drug from the market. The aim of this paper was to develop and validate an HPLC-UV method for the quantification of 4′-hydroxydiclofenac as a CYP2C9 metabolite using salicylic acid as an inhibitor in rat liver microsomes. A CYP2C9 assay was developed and validated on the reversed phase C₁₈ column (SUPELCO 25 cm × 4.6 mm × 5 µm) using a low-pressure gradient elution programming at T = 30 °C, a wavelength of 282 nm, and a flow rate of 1 mL/min. 4′-hydroxydiclofenac demonstrated a good linearity (R² > 0.99), good reproducibility, low detection, and quantitation limit, and the inter and intra-day precision met the ICH guidelines (<15%). 4′-hydroxydiclofenac was stable for three days and showed an acceptable accuracy and recovery (80–120%) within the ICH guidelines in a rat liver microsome sample. This method will be beneficial for future applications of the in vitro inhibitory effect of salicylic acid on the CYP2C9 enzyme activity in rat microsomes and the in vivo administration of salicylic acid in clinical trials.     

Enthalpic and Liquid-Phase Adsorption Study of Toluene–Cyclohexane and Toluene–Hexane Binary Systems on Modified Activated Carbons

The liquid-phase adsorption of toluene in cyclohexane and hexane solutions on modified activated carbons was evaluated; the energy involved in the interaction between these solutions and the solids was determined by immersion enthalpies of pure solvents and their mixtures, and the contribution of the system constituents was calculated by differential enthalpies. The thermal treatment generated modifications that favored adsorption and interaction with the evaluated solutions, since it increased the textural parameters and the basic character of the samples. Cyclohexane could create greater competition with the adsorption sites compared to hexane, but it favored the increase in adsorption capacities (0.416 to 1.026 mmol g⁻¹) and the interactions with the solid evaluated through the immersion enthalpies. The immersion enthalpies of pure solvents (−16.36 to −112.7 J g⁻¹) and mixtures (−25.65 to −104.34 J g⁻¹) had exothermic behaviors that were decreasing due to the possible displacement of solvent molecules when increasing the solute concentration in the mixtures. The differential enthalpies for toluene were negative (−18.63 to −2.14 J), mainly due to the π–π interaction with the solid, while those of the solvent–solid component tended to be positive values (−4.25 to 55.97 J) due to the displacement of the solvent molecules by those of toluene.     

Interaction Between Surfactin and Bovine Serum Albumin

The interaction of surfactin, a typical biosurfactant, with bovine serum albumin (BSA) was investigated by surface tension, fluorescence, freeze-fractured transmission electron microscopy (FF-TEM) and circular dichroism (CD) measurements. The surface tension curves of pure surfactin solution and surfactin/BSA solutions have different phenomena, where two obvious inflections determined as the critical aggregation concentration (cac) and the critical micelle concentration (cmc) appear for surfactin/BSA solutions. The higher BSA concentration, the higher cac and cmc values for surfactin/BSA solution. Fluorescence spectra show that the structure change of BSA is dependent on both surfactin and BSA concentration. The micropolarity, FF-TEM and CD results further demonstrate the interaction between BSA and surfactin. The excess free energy (ΔG⁰) of surfactin/BSA interactions have been obtained as ?6.13 and 5.32 kJ/mol for 1.0 × 10^?6 and 3.8 × 10^?6 mol/L BSA concentration, respectively. The binding ratio (R) determined for surfactin/BSA systems are higher than that reported for dirhamnolipid to BSA. Above all, it can be concluded that the hydrophobic interaction and the hydrogen bonds between surfactin and BSA play the key role for the high binding ratio for surfactin to BAS.     

Studies of (3-mercaptopropyl)trimethoxylsilane and bis(trimethoxysilyl)ethane sol–gel coating on copper and aluminum

Berbamine, a naturally occurring isoquinoline alkaloid extracted from Berberis sp., is the active constituent of some Chinese herbal medicines and exhibits a variety of pharmacological activities. The effects of berbamine on the structure of bovine serum albumin (BSA) were investigated by circular dichroism, fluorescence and absorption spectroscopy under physiological conditions. Berbamine caused a static quenching of the intrinsic fluorescence of BSA, and the quenching data were analyzed by application of the Stern–Volmer equation. There was a single primary berbamine-binding site on BSA with a binding constant of 2.577 × 10⁴ L mol⁻¹ at 298 K. The thermodynamic parameters, enthalpy change (ΔH⁰) and entropy change (ΔS⁰) for the reaction were −76.5 kJ mol⁻¹ and −173.4 J mol⁻¹ K⁻¹ according to the van’t Hoff equation. The results showed that the hydrogen bond and van der Waals interaction were the predominant forces in the binding process. Competitive experiments revealed a displacement of warfarin by berbamine, indicating that the binding site was located at Drug sites I. The distance r between the donor (BSA) and the acceptor (berbamine) was obtained according to the Förster non-radiation energy transfer theory. The results of three-dimensional fluorescence spectra, UV–vis absorption difference spectra and circular dichroism of BSA in the presence of berbamine showed that the conformation of BSA was changed. The results provide a quantitative understanding of the effect of berbamine on the structure of bovine serum albumin, providing a useful guideline for further drug design.