Tocainide analogues binding to human serum albumin: A HPLAC and circular dichroism study

A series of synthesised tocainide analogues were characterized for their human serum albumin (HSA) binding, using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). The synthesis and physico-chemical characterization of compounds 7a–7d is reported here. For the HPLAC investigation HSA was covalently immobilized to the silica matrix of the HPLC column, using an anchoring procedure, which allows the binding properties of the protein to be maintained. The HSA-based column was used for getting information on the high affinity binding sites of the tocainide analogues to HSA. According to the displacement chromatography approach, the retentions of the analytes were determined in the absence and in the presence of increasing concentrations of competitors known to bind to specific binding sites on the protein. The same system, drug/protein, was investigated in solution by CD.     

Interaction of isoliquiritigenin with bovine serum albumin studied by fluorescence quenching method

Interaction of ioliquiritigenin (ISL), which is the main active component of a commonly used traditional Chinese medicine (TCM) Glycyrrhiza uralensis Fisch. with bovine serum albumin (BSA) has been investigated. The quenching mechanism of fluorescence of bovine serum albumin by ISL was discussed. The binding sites number n and apparent binding constant K were measured by fluorescence quenching method. The thermodynamic parameters ΔH⁰, ΔG⁰, ΔS⁰ at different temperatures were calculated. The distance r between donor (bovine serum albumin) and acceptor (ISL) was obtained according to Förster theory of non-radiation energy transfer. The results of synchronous fluorescence spectra and UV-vis absorption spectra show that the conformation of bovine serum albumin has been changed.     

Binding interaction of indomethacin with human serum albumin

The interaction between indomethacin and human serum albumin (HSA) was investigated by fluorescence quenching technique and UV-vis absorption spectroscopy. The results of fluorescence titration revealed that indomethacin, strongly quench the intrinsic fluorescence of HSA by static quenching and nonradiative energy transfer. The binding site number n and the apparent binding constant K(A), were calculated using linear and nonlinear fit to the experimental data. The distance r between donor (HSA) and acceptor (indomethacin) was obtained according to fluorescence resonance energy transfer (FRET). The study suggests that the donor and the acceptor are bound at different locations but within the quenching distance.     

Interactions of human serum albumin with meloxicam: Characterization of binding site

Human serum albumin (HSA) is the most prominent protein in plasma. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme, bilirubin and drugs. Here, we report the effect of new generation, non-steroidal anti-inflammatory drug (NSAID) meloxicam on the albumin conformation and ligand binding. In the present work the interaction of meloxicam with HSA in aqueous solution at physiological pH has been investigated through circular dichroism and fluorescence spectroscopy. The strong quenching of the fluorescence clearly indicated that the binding of the drug to HSA changed the microenvironment of tryptophan residue and the tertiary structure of HSA. This was confirmed by the destabilization of the warfarin binding site. CD and fluorescence spectroscopic results showed marked reductions (about 40% decrease in the CD Cotton effect intensity, and ∼15% decrease of the fluorescence intensity) in the affinity of albumin for bilirubin upon meloxicam binding. The strong inhibition of warfarin and ANS bound to protein after meloxicam modification compared with aspirin confirms that the binding site of both drugs is not the same.     

花菁探针荧光增强法测定血清蛋白的研究

基于蛋白质对花菁染料的荧光增强效应,以水溶性碳菁染料（1,1′-丙磺酸-3,3,3′,3′-四甲基吲哚三次甲基碳菁-5,5′-二磺酸钾）（STEID）为探针,建立了蛋白质荧光检测体系。考察了pH、染料浓度和离子等影响,在最优条件下,蛋白质对该碳菁染料具有明显的增强作用,荧光强度与蛋白质浓度成良好的线性关系,牛血清蛋白BSA线性响应范围为0.20~10.0μg.mL-1,检测灵敏度（3σ/K）为0.07μg.mL-1。测定了BSA血清合成样品,回收率88.5%~98.0%。     

Studies on interaction between Vitamin B12 and human serum albumin

The binding reaction between Vitamin B12 (B12, cyanocobalamin) and human serum albumin (HSA) was investigated by fluorescence quenching, UV–vis absorption and circular dichroism (CD) spectroscopy. Under simulative physiological conditions, fluorescence quenching data revealed that the quenching constants (K_sv) are 3.99 × 10⁴, 4.33 × 10⁴, 4.76 × 10⁴ and 5.16 × 10⁴ M⁻¹ at 292, 298, 304 and 310 K, respectively. The number of binding sites, n is almost constant around 1.0. On the basis of the results of fluorescence quenching the mechanism of the interaction of B12 with HSA has been found to be a dynamic quenching procedure. Thermodynamic parameters ΔH^Θ = −13.38 kJ mol⁻¹, ΔS^Θ = 66.73 J mol⁻¹ K⁻¹ were calculated based on the binding constant. These suggested that the binding reaction was enthalpy and entropy driven, and the electrostatic interaction played major role in stabilizing the reversible complex. The binding distance r = 5.5 nm between HSA and B12 was obtained according to Förster theory of energy transfer. The effect of B12 on the conformation of HSA was analyzed by synchronous fluorescence and CD spectroscopy. Synchronous spectra indicated that the polarity around the tryptophan (Trp214) residues of HSA was decreased and its hydrophobicity was increased; however, the α-helix content of the protein was predominant in the secondary structure but the CD spectra indicated that B12 induced minor conformational changes of HSA.     

Drug-Biomacromolecule Interaction XII: Comparative binding study of sulfaethidole to bovine serum albumin by equilibrium dialysis,fluorescence probe technique,uv difference spectrophotometry and circular dichroism

Binding of sulfaethidole to bovine serum albumin was studied by equilibrium dialysis, fluorescence probe technique, uv difference spectrophotometry and circular dichroism. Equilibrium dialysis method enabled us to estimate the total number of drug binding sites of albumin molecule. For sulfaethidole, albumin had 6 primary and 40 secondary binding sites. The primary and secondary binding constants were 0.9×10⁵ M⁻¹ and 0.2×10⁶ M⁻¹, respectively. 1-Anilino-8-naphthalenesulfonate (ANS) and 2-(4′-hydroxylbenzeneazo)-benzoic acid (HBAB) were used as the fluorescence probe and the uv spectrophotometric probe, respectively. In fluorescence probe technique, results indicated that the number of higher affinity drug binding site of albumin was 1 and the number of lower affinity drug binding sites of albumin was 3, and the primary and secondary drug binding constants for bovine serum albumin were 2.15×10⁵ M⁻¹ and 1.04×10⁵ M⁻¹, respectively. In uv difference spectrophotometry, binding sites were 3 and binding constant was 1.88×10⁵ M⁻¹. The above results suggest that several different methods should be used in ompensation for insufficient information about drug binding to albumin molecule given by only one method.     

阿柔比星与人血白蛋白相互作用的特点

目的研究阿柔比星与人血清白蛋白的相互作用特点。方法用荧光猝灭光谱及同步荧光光谱技术测定在不同温度下阿柔比星对人血清白蛋白荧光的猝灭规律。结果阿柔比星对人血清白蛋白荧光呈规律性猝灭,17℃时的n为1.11,K为6.21×104,37℃时的n为1.12,K为6.41×104。阿柔比星使白蛋白的同步光谱中最大发射峰红移。结论阿柔比星与人血清白蛋白只有一个结合位点,表现为疏水作用,两者结合使白蛋白的极性略有增加。     

吖啶药物与血清蛋白的相互作用研究

应用荧光谱法,研究（N-（N-（2-（4-吗啉）乙胺）-4-酰胺吖啶）-α-丙氨酸（MACA）与血清白蛋白（HSA）的相互作用,确定MACA-HSA的静态荧光猝灭机制和疏水力相互作用,系统考察MACA与HSA的结合常数、结合位点数、热力学函数,两者在不同温度下的结合常数分别为2.51×10^5（298K）、1.78×10^5（308K）、1.32×10^5（318K）;结合位点数分别为1.05、1.08、1.10,MACA和HSA结合作用的ΔH和ΔS分别为-25.39kJ·mol^-1和18.20kJ·mol^-1。     

Study of the interaction between fluoroquinolones and bovine serum albumin

The mechanism of interaction between norfloxacin (NRF) and ciprofloxacin (CPF) with bovine serum albumin has been investigated using circular dichroism, fluorescence and absorption spectroscopy. The quenching mechanism of fluorescence of bovine serum albumin by fluoroquinolones was discussed. The binding sites number n and apparent binding constant K were measured by fluorescence quenching method. The thermodynamic parameters obtained from data at different temperatures were calculated. The distance r between donor (bovine serum albumin) and acceptor (fluoroquinolones) was obtained according to Forster theory of non-radiation energy transfer. The effect of common ions on binding constant was also investigated. The results of synchronous fluorescence spectra, UV–vis absorption spectra and circular dichroism of BSA in presence of fluoroquinolones show that the conformation of bovine serum albumin changed.     

Study of the interaction between mercury (II) and bovine serum albumin by spectroscopic methods

Mercury is a significant environmental pollutant that originates from industry. Mercury will bind with albumin and destroy biological functions in humans if it enters the blood. In this paper, the interaction between mercury (II) and bovine serum albumin (BSA) was investigated in vitro by fluorescence, UV–Vis absorption and circular dichroism (CD) under simulated physiological conditions. This study proves that the probable quenching mechanism of BSA by mercury (II) was mainly static quenching due to the formation of a mercury (II)–BSA complex. The quenching constant K_a and the corresponding thermodynamic parameters (ΔH, ΔS and ΔG) at four different temperatures were calculated by a modified Stern–Volmer equation and the van’t Hoff equation, respectively. The results revealed that the interaction between mercury (II) and BSA was mainly enthalpy-driven and that hydrogen bonding and van der Waals forces played a major role in the reaction. The obtained data for binding sites of n approximately equal to 1 indicated that there was a single class of binding site for the BSA with mercury (II). The value of the distance r (3.55 nm), determined by Föster's non-radioactive energy transfer theory, suggested that the energy transfer from BSA to mercury (II) occurred with a high probability. The conformational investigation from synchronous fluorescence, CD spectroscopy and three-dimensional fluorescence showed that the presence of mercury (II) resulted in micro-environmental and conformational changes of the BSA molecules, which may be responsible for the toxicity of mercury (II) in vivo.     

Interaction of anthracycline disaccharide with human serum albumin: investigation by fluorescence spectroscopic technique and modeling studies

Anthracyclines are considered to be some of the most effective anticancer drugs for cancer therapy. However, drug resistance and cardiotoxicity of anthracyclines limit their clinical application. An 3′-azido disaccharide analogue of daunorubicin, 7-[4-O-(2,6-dideoxy-3-O-methyl-α-l-arabino-hexopyranosyl)-3-azido-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone (ADNR-3), was shown to exhibit 10-fold better activity than parent compound daunorubicin against the drug-resistant cells and completely overcomes the drug resistance with same IC₅₀ in both drug-resistant and drug-sensitive cells. In this paper, the interactions between ADNR-3 and human serum albumin (HSA) have been studied by spectroscopic techniques. By the analysis of fluorescence spectrum and fluorescence intensity, it was observed that the ADNR-3 has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The association constants of ADNR-3 with HSA were determined at different temperatures based on fluorescence quenching results. The negative ΔH and positive ΔS values in case of ADNR-3–HSA complexes showed that both hydrogen bonds and hydrophobic interactions play a role in the binding of ADNR-3 to HSA. Furthermore, synchronous fluorescence spectroscopy data and UV–vis absorbance spectra have suggested that the association between ADNR-3 and HSA changed the molecular conformation of HSA and the hydrophobic interactions play a major role in ADNR-3–HSA association. Moreover, the study of computational modeling indicated that ADNR-3 could bind to the site I of HSA and hydrophobic interaction was the major acting force for the second type of binding site, which was in agreement with the thermodynamic analysis. The distance, r, between donor (HSA) and acceptor (ADNR-3) was obtained according to the Förster's theory of non-radiation energy transfer. In addition, the effects of common ions on the binding constants of ADNR-3–HSA complexes were also investigated.     

Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes

The interaction between triprolidine hydrochloride (TRP) to serum albumins viz. bovine serum albumin (BSA) and human serum albumin (HSA) has been studied by spectroscopic methods. The experimental results revealed the static quenching mechanism in the interaction of TRP with protein. The number of binding sites close to unity for both TRP-BSA and TRP-HSA indicated the presence of single class of binding site for the drug in protein. The binding constant values of TRP-BSA and TRP-HSA were observed to be 4.75 ± 0.018 × 10(3) and 2.42 ± 0.024 × 10(4)M(-1) at 294 K, respectively. Thermodynamic parameters indicated that the hydrogen bond and van der Waals forces played the major role in the binding of TRP to proteins. The distance of separation between the serum albumin and TRP was obtained from the F?rster's theory of non-radioactive energy transfer. The metal ions viz., K(+), Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Zn(2+) were found to influence the binding of the drug to protein. Displacement experiments indicated the binding of TRP to Sudlow's site I on both BSA and HSA. The CD, 3D fluorescence spectra and FT-IR spectral results revealed the changes in the secondary structure of protein upon interaction with TRP.     

Inhibition of drug binding to human serum albumin by cholecystographic agents

The binding of two cholecystographic agents to human serum albumin (HSA) was evaluated by means of two different complementary methodologies. In particular, the inhibition of drug HSA binding caused by iopanoic- and iophenoxic-acid was investigated by circular dichroism (CD) and resonant mirror (RM) optical biosensor techniques. The CD study allowed to obtain information both on the cholecystographic agent binding site and on the effect of the binding on the protein conformation. Iopanoic acid (IOP), a drug potentially useful for thyrotoxic disorders, resulted a direct competitor for ligands that selectively bind to site II, in agreement to literature data. No definite evidence was obtained for the highest affinity binding site of iophenoxic acid (IOPH), however, this diagnostic tool markedly affected the binding of ligands to the most characterized high affinity sites on HSA, namely sites I, II and III. Binding parameters were obtained by optical biosensor analysis: K(D) values were 3.6 x 10(-7) and 2.8 x 10(-8) M for IOP and IOPH, respectively.     

Binding of Sudan II and Sudan IV to bovine serum albumin: Comparison studies

In this paper, we report the interaction of Sudan II and Sudan IV to bovine serum albumin (BSA). Structural analysis showed that both Sudan II and Sudan IV interact mainly with BSA at the hydrophobic pocket and via Van der Waals forces. The number of bound Sudan molecule for each protein molecule was approximately 1. The overall binding constants at 293 K (20 °C) estimated for Sudan II and Sudan IV were 1.22 × 10⁴ M⁻¹ and 1.48 × 10⁴ M⁻¹, respectively. BSA backbone structure was damaged by the dyes with more severe phenomenon observed for Sudan IV. For two Sudan dyes with the same concentration, Sudan IV could cause more alterations on CD spectra of BSA with slight decrease of α-helical content and increase of β-sheet content, suggesting a partial protein unfolding.     

Spectroscopic studies on the interaction between silicotungstic acid and bovine serum albumin

The interaction between silicotungstic acid and bovine serum albumin (BSA) was investigated using fluorescence and UV/vis. The experimental results showed that the fluorescence quenching of BSA by silicotungstic acid is a result of the formation of SiW–BSA complex; static quenching and non-radiative energy transferring were confirmed to result in the fluorescence quenching. The binding site number n, apparent binding constant K_A and corresponding thermodynamic parameters were measured at different temperatures. The process of binding SiW molecule on BSA was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic interaction force plays a major role in stabilizing the complex. The effect of silicotungstic acid on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy.     

大黄素甲醚和牛血清蛋白相互作用的电化学性质研究

在pH4.0的Britton-Robinson(B-R)缓冲体系中,应用循环伏安法和示差脉冲伏安法对大黄素甲醚与牛血清白蛋白(BSA)相互作用的电化学性质进行研究。结果表明,二者结合生成了一种非电活性的超分子化合物,同时对结合反应的机理进行了探讨。BSA的存在导致大黄素甲醚氧化还原峰电流降低,峰电位基本不变,峰电流的下降值同所加入的BSA浓度在一定范围内呈线性关系。线性范围5.0×10-6~1.0×10-7mol/L,检出限3×10-7mol/L。     

吲哚菁类探针与血清蛋白的相互作用研究

目的应用荧光光谱法,研究了新型水溶性吲哚基同型聚合体菁类探针I与牛血清蛋白（BSA）的相互作用。方法探索吲哚探针I-BSA的静态荧光猝灭机制和静电相互作用,系统考察了吲哚探针I与BSA的结合常数、结合位点数、热力学函数。结果确定了两者在不同温度下的结合常数分别为3.72×10^5（293K）、3.59×10^5（298K）、3.40×10^5（303K）;结合位点数分别为1.02、0.99、0.98,吲哚探针I和BSA结合作用的ΔH,ΔS和ΔG分别为-2.242kJ.mol^-1,98.80kJ.mol^-1和-31.69kJ.mol^-1（298K）。结论吲哚探针I与牛血清蛋白之间的结合作用力主要为静电作用。     

Study on the interaction of bioactive compound S-allyl cysteine from garlic with serum albumin

Multispectroscopic techniques were used to investigate the interaction of S-allyl cysteine (SAC) from garlic with human serum albumin (HSA). UV–Vis absorption measurements prove the formation of the HSA–SAC complex. An analysis of fluorescence spectra revealed that in the presence of SAC, the quenching mechanism of HSA is considered static. The quenching rate constant K_q, K_SV, and the binding constant K_A were estimated. According to the Van’t Hoff equation, the thermodynamic parameters enthalpy change (ΔH) and entropy change (ΔS) were calculated to be −1.00 × 10⁵ J/mol and −255 J/mol/K, respectively. These indicate that hydrogen bonds and van der Waals forces are the major forces between SAC and HSA. The changes in the secondary structure of HSA, which was induced by SAC, were determined by circular dichroism spectroscopy. Energy transfer was confirmed and the distance between donor and acceptor was calculated to be 2.83 nm.     

Circular dichroism studies on the inhibiting effect of oleic acid on the binding of diazepam to human serum albumin

The effect of a free fatty acid (oleic acid) on the binding of a benzodiazepine derivative (diazepam) to human serum albumin (HSA)¹ has been studied using the technique of circular dichroism. Both qualitative and quantitative results suggest that oleic acid significantly affects the binding of diazepam, even at low molar ratios to albumin (below 1:1). It is suggested that the displacement of bound diazepam occurs primarily through an allosteric mechanism.