Multiple spectroscopic studies on the interaction between olaquindox,a feed additive,and bovine serum albumin

The interaction between olaquindox (OLA) and bovine serum albumin (BSA) was investigated using fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopy. The results showed that the fluorescence quenching of BSA by OLA was a static quenching process induced by the formation of OLA–BSA complex. The binding constant of OLA–BSA complex was calculated to be 1.299 × 10⁴ L mol⁻¹ (293 K). The negative values of ΔH⁰ and ΔS⁰ indicated that hydrogen bond and van der Waals interactions played major roles in stabilizing the complex. Site probe competition experiments and number of binding sites (n) revealed that OLA could bind to site I in subdomain IIA of BSA, and the binding distance (r) was evaluated to be 3.643 nm according to Förster’s non-radiative energy transfer theory. The results of CD and three-dimensional fluorescence spectra suggested some conformational changes of BSA after OLA binding.     

Study on the interaction between salicylic acid and catalase by spectroscopic methods

The interaction between catalase (CAT) and salicylic acid (SA) was studied by fluorescence and UV–vis spectroscopic techniques. The quenching mechanism of fluorescence of BSA by CAT was discussed to be a static quenching procedure. The number of binding sites n and apparent binding constant K was measured by fluorescence quenching method. The thermodynamics parameter ΔH, ΔG, ΔS were calculated. The results indicate the binding reaction was both entropy-driven and the enthalpy-driven, and the hydrogen bond and van der Waals force played major role in the binding reaction. The binding sites of SA with CAT was investigated to be approached the microenvironment of Trp by the synchronous fluorescence spectrometry. The distance r between donor (CAT) and acceptor (SA) was obtained according to Förster theory of non-radioactive energy transfer.     

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.     

Study of the interaction of an anticancer drug with human and bovine serum albumin: spectroscopic approach

The interactions between gemcitabine hydrochloride (GEM) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by spectroscopic techniques. By the analysis of fluorescence spectrum and fluorescence intensity, it was observed that the GEM has a strong ability to quench the intrinsic fluorescence of both BSA and HSA through a static quenching procedure. The association constants of GEM with BSA and HSA were determined at different temperatures based on fluorescence quenching results. The negative ΔH° and positive ΔS° values in case of GEM–BSA and GEM–HSA complexes showed that both hydrogen bonds and hydrophobic interactions play a role in the binding of GEM to BSA or HSA. Experimental results showed that the binding of GEM to BSA or HSA induced conformational changes in BSA and HSA. From the quantitative analysis data of CD spectra, the α-helix of 57.58% and 34.82% in free BSA and free HSA decreased to 40.82% and 29.84% in BSA–GEM and HSA–GEM complexes, respectively, and hence confirmed that the secondary structure of protein was altered by GEM. The interactions of BSA and HSA with GEM were also confirmed by UV absorption spectra. The distance, r, between donor (BSA or HSA) and acceptor (GEM) was obtained according to the Förster's theory of non-radiation energy transfer. The effects of common ions on the binding constants of both BSA–GEM and HSA–GEM complexes were also investigated.     

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.     

Molecular interaction of tea catechin with bovine β-lactoglobulin: A spectroscopic and in silico studies

Polyphenols has attained pronounced attention due to their beneficial values of health and found to prevent several chronic diseases. Here, we elucidated binding mechanism between frequently consumed polyphenol “tea catechin” and milk protein bovine beta-lactoglobulin (β-Lg). We investigated the conformational changes of β-Lg due to interaction with catechin using spectroscopic and in silico studies. Fluorescence quenching data (Stern-Volmer quenching constant) revealed that β-Lg interacted with catechin via dynamic quenching. Thermodynamic data revealed that the interaction between β-Lg and catechin is endothermic and spontaneously interacted mainly through hydrophobic interactions. The UV-Vis absorption and far-UV circular dichroism (CD) spectroscopy exhibited that the tertiary as well as secondary structure of β-Lg distorted after interaction with catechin. Molecular docking and simulation studies also confirm that catechin binds at the central cavity of β-Lg with high affinity (~10⁵ M⁻¹) and hydrophobic interactions play significant role in the formation of a stable β-Lg-catechin complex.     

Mechanistic interaction study of 5,6-Dichloro-2-[2-(pyridin-2-yl)ethyl]isoindoline-1,3-dione with bovine serum albumin by spectroscopic and molecular docking approaches

A synthesized and promising biologically hypoglycemic compound 5,6-Dichloro-2-[2-(pyridin-2-yl)ethyl]isoindoline-1,3-dione (5e) was studied for its binding to a model protein (bovine serum albumin; BSA) by spectroscopic and molecular simulation approaches. Fluorescence studies revealed that 5e quenched BSA’s intrinsic fluorescence by static quenching. The experiments were performed at three different temperatures and the quenching constants and binding constants were evaluated. Stern-Volmer constant (K_sv) values decreased from 1.36?×?10⁴ to 1.20?×?10⁴ as the temperature increased suggesting static quenching involvement in the interaction. Decreased binding constants from 1.70?×?10⁴ to 4.57?×?10³ at higher temperatures indicated instability of the complex at rising temperatures. Site I (subdomain IIA) of BSA was found to interact with 5e. The thermodynamic results showed the binding interaction was spontaneous and enthalpy driven. The secondary structure alterations in BSA due to interaction with 5e were studied by UV–visible, synchronous fluorescence, and three-dimensional fluorescence spectra. The results indicate the 5e binds effectively to the BSA and thus, this study can be useful in further exploring the pharmacokinetics and pharmacodynamics of 5e.     

Biophysical study on the interaction of etomidate and the carrier protein in vitro

Etomidate is a unique drug used for induction of general anesthesia and sedation, and is usually used through intravenous injection clinically. Before targeting to the receptor, etomidate binds proteins in blood when it comes into veins. Thus to study the interaction of etomidate and serum albumin would be of great toxicological and pharmacological importance. In this study, the interaction between etomidate and human serum albumin (HSA) was studied using fluorescence spectroscopy, UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) spectroscopy, site maker displacement and molecular modeling methods. Investigations of the binding constant (K?=?3.55?×?10^5?M^?1, 295?K), the number of binding sites (n?=?1.16), thermodynamic parameters (ΔG?=?3.13?×?10^4?J·mol^?1, ΔS?=?364?J·mol^?1·K^?1 and ΔH?=??6.85?×?10^5?J·mol^?1) for the reaction and changes to the binding sites and conformation in HSA in response to etomidate were presented. Results show that etomidate can bind HSA tightly through electrostatic forces, and the protein skeleton conformation and secondary structure changes thereby. This is the first spectroscopic report for etomidate–HSA interactions which illustrates the complex nature of this subject.     

Complexation of cox-2 inhibitors with bovine serum albumin: Interaction mechanism

Mechanism of interaction of six cox-2 inhibitors – celecoxib, valdecoxib, etoricoxib, parecoxib sodium, meloxicam and nimesulide – with bovine serum albumin (BSA) was studied using fluorescence spectroscopic technique. Results were discussed in terms of the binding parameters, thermodynamics of the binding process, the nature of forces involved in the interaction and the fluorescence quenching mechanism involved. Association constants were of the order of 10⁴–10⁵ for various drugs. Binding affinity varied with the nature of drug. Nature of forces involved in the interaction could be predicted from the thermodynamic parameters for the binding. Meloxicam and nimesulide shared common sites with hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS) on BSA molecule. Stern-Volmer analysis of the quenching data indicated that both tryptophan residues of BSA are fully accessible to the drugs and predominantly static quenching mechanism is involved in the interaction.     

Interaction of gallic acid with trypsin analyzed by spectroscopy

The interactions between trypsin and gallic acid (GA) were investigated by means of fluorescence spectroscopy, UV-vis absorption spectroscopy, resonance light scattering (RLS) spectroscopy, synchronous fluorescence spectroscopy, and enzymatic inhibition assay. It was found that GA can cause the fluorescence quenching of trypsin during the process of formation of GA-trypsin complex, resulting in inhibition of trypsin activity (IC₅₀ = 3.9 × 10⁻⁶ mol/L). The fluorescence spectroscopic data showed that the quenching efficiency can reach about 80%. The binding constants were 1.9371 × 10⁴ L/mol, 1.8192 × 10⁴ L/mol, and 1.7465 × 10⁴ L/mol at three temperatures, respectively. The thermodynamic parameters revealed that hydrogen bonds, van der Waals, hydrophobic, and electrostatic interactions were involved in the binding process of GA to trypsin. Molecular modeling studies illustrated a specific display of binding information and explained most of the experiment phenomena. The microenvironments of tryptophan and tyrosine residue in trypsin were changed by the GA. Results indicated that GA was a strong quencher and inhibitor of trypsin.     

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.     

牡荆苷与溶菌酶相互作用的荧光光谱学研究

目的在模拟人体生理条件下,研究牡荆苷与溶菌酶的相互作用的光谱学特征。方法利用同步荧光光谱法和荧光光谱法研究牡荆苷与溶菌酶的相互作用机制、作用力类型,考察牡荆苷对溶菌酶构象的影响。结果牡荆苷对溶菌酶的荧光猝灭机制为静态猝灭,牡荆苷与溶菌酶的结合常数、结合位点数分别为6.73×10~5 L/mol、1.12,其作用力以疏水作用为主。同步荧光光谱法测定的结合位点靠近色氨酸,并使色氨酸的疏水性增强。结论牡荆苷与溶菌酶结合并改变溶菌酶的构象,共存金属离子对牡荆苷与溶菌酶的相互作用有一定的影响。     

Interaction of bioactive coomassie brilliant blue g with protein: insights from spectroscopic methods

The binding of coomassie brilliant blue G (CBB) to bovine serum albumin (BSA) was investigated under simulative physiological conditions employing different optical spectroscopic techniques viz., fluorescence emission, UV–visible absorption and FTIR. Fluorescence quenching data obtained at different temperatures suggested the presence of dynamic type of quenching mechanism. The binding constant of CBB-BSA and the number of binding sites (n) for CBB in BSA were calculated and found to be 4.20 × 10⁴ M⁻¹ and 0.96 respectively, at 302 K. The value of n close to unity indicated that the protein has a single class of binding sites for CBB. The thermodynamic parameters revealed that the hydrophobic forces played a major role in the interaction of CBB to BSA. The distance between the CBB and protein was calculated using the theory of Föster’s Resonance Energy Transfer (FRET). The conformational change in the secondary structure of BSA upon interaction with dye was investigated by synchronous fluorescence and FTIR techniques. Competitive binding studies were also carried out to know the location of binding of CBB on BSA.     

Molecular spectroscopic studies on the interaction between Ractopamine and bovine serum albumin

To investigate the interaction between Ractopamine (RAC), an animal growth promoter, and bovine serum albumin (BSA), three spectroscopic approaches (fluorescence, UV–vis and FT-IR) and three different experiments (two mole-ratio and a Job's methods) were used to monitor the biological kinetic interaction procedure. The Stern–Volmer quenching constants K_SV, the binding constants K_a, and the number of binding sites n at 298, 301 and 304 K were evaluated by molecular spectroscopic approaches. The values of enthalpy (−13.47 kJ mol⁻¹) and entropy (78.39 J mol⁻¹ K⁻¹) in the reaction indicated that RAC bound to BSA mainly by electrostatic and hydrophobic interaction. The site markers competitive experiments indicated that the binding of RAC to BSA primarily took place in site I. The spectra data matrix was further investigated with multivariate curve resolution-alternating least squares (MCR-ALS), and the concentration profiles and the pure spectra for three species (BSA, RAC and RAC-BSA) existed in the kinetic interaction procedure, as well as the apparent equilibrium constants, were obtained.     

Exploring the mechanism of interaction between 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one and human serum albumin: Spectroscopic, calorimetric and molecular modeling studies

In this paper, binding interaction of 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (EMMD) with human serum albumin (HSA) under physiological conditions was investigated by using spectroscopy, isothermal titration calorimetry (ITC) and molecular modeling techniques. The results of spectroscopic studies suggested that EMMD have a strong ability to quench the intrinsic fluorescence of HSA through static quenching procedure. ITC investigations indicated that drug-protein complex was stabilized by hydrophobic forces and hydrogen bonds, which was consistent with the results of molecular modeling studies. Competitive experiments indicated the displacement of warfarin by EMMD, which revealed that the binding site of EMMD to HSA was located at subdomain IIA.     

Probing the binding of morin to human serum albumin by optical spectroscopy

Morin [2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one], a member of flavonols, is an important bioactive compound by interacting with nucleic acids, enzymes and protein. Its binding to human serum albumin was investigated by fluorescence quenching, fluorescence anisotropy, and UV–vis absorbance under the simulative physiological condition. Fluorescence quenching data show that the interaction of morin with HSA forms a non-fluorescent complex with the binding constants of 1.394 × 10⁵, 1.489 × 10⁵, 1.609 × 10⁵ and 1.717 × 10⁵ M⁻¹ at 292, 298, 303 and 310 K, respectively. The thermodynamics parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be 8.97 kJ mol⁻¹ and 129.15 J mol⁻¹ K⁻¹ via van’t Hoff equation. From the spectroscopic results and thermodynamics parameters, it is observed that van der Waals and hydrogen bonds are predominant intermolecular forces when forming the complex. The distance r = 4.25 nm between donor (Trp214) and accepter (morin) was estimated based on the Förster theory of non-radiative energy transfer. The red shift of UV–vis absorbance shows that morin is bound to several amino acids on the hydrophobic pocket of HSA. Moreover, the competitive probes, such as warfarin and ibuprofen (site I and II probes, respectively), reveal that the binding location of morin to HSA in the site I of the hydrophobic pocket, which corresponds to the results of UV–vis absorbance, while morin also binds other lower affinity binding sites on HSA from the fluorescence anisotropy spectroscopy.     

荧光光谱法研究卡铂与牛血清白蛋白的相互作用

目的研究卡铂与牛血清白蛋白(BSA)在人体生理条件下的相互作用。方法采用荧光光谱法研究卡铂与BSA的荧光猝灭机制、结合位点数、结合常数;利用热力学参数考察其作用力类型;采用同步荧光光谱法探讨卡铂对BSA构象的影响。结果卡铂与BSA形成1∶1的复合物引起BSA的荧光猝灭,其猝灭类型为静态猝灭。卡铂与BSA结合位点数为9.81×103 mol/L,两者以疏水作用为主。卡铂与BSA相互作用使色氨酸残基所处的微环境发生改变。结论卡铂与BSA相互作用形成复合物,并改变BSA的构象。     

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.     

Optical Analysis of Zinc Oxide Quantum Dots with Bovine Serum Albumin and Bovine Hemoglobin

Quantum dots (QDs) are widely used in medical, industrial, and household applications owing to their excellent biological property. For its wide medical application, the biocompatibility of QDs is an important aspect of research. The aim of the present study was to synthesize zinc oxide quantum dots (ZnO-QDs) and to investigate the interaction with bovine serum albumin (BSA) and bovine hemoglobin (BHb) using fluorescence quenching method and circular dichroism (CD). The study suggests that the electrostatic force of attraction favors the adsorption of BSA onto ZnO-QDs. The fluorescence quenching of BSA and BHb using QD indicates the formation of QDs-BSA complexes. The CD spectra also showed the changes in secondary structure of proteins by interacting with QDs.     

Oxidation-reduction state of free NADP+ during mixed-function oxidation in perfused rat livers--Evaluation of the assumptions of near equilibrium by comparisons of surface fluorescence changes and calculated NADP+:NADPH ratios.

Changes in cellular pyridine nucleotide and flavoprotein oxidation-reduction states associated with mixed-function oxidation of p-nitroanisole, hexobarbital and aminopyrine by perfused rat livers were studied. Surface fluorescence techniques were compared with NAD(P)⁺/NAD(P)H ratios calculated from substrates assumed to be in near equilibrium with various dehydrogenases in freeze-clamped liver samples. p-Nitroanisole and p-nitrophenol caused a large decrease in pyridine nucleotide (366 → 450 nm) fluorescence as a result of fluorescence quenching. This decrease, therefore, did not reflect oxidation of pyridine nucleotides. Moreover, p-nitroanisole infusion decreased the free NADP⁺:NADPH ratios calculated from malic enzyme and isocitrate dehydrogenase. Hexobarbital, which did not produce fluorescence quenching, caused an oxidation in pyridine nucleotides as indicated by both a decrease in surface fluorescence and an increase in the calculated NADP⁺/NADPH ratio. These data indicate that free NADP⁺/NADPH ratios calculated from substrates, which are assumed to be in near equilibrium with NADPH-generating enzymes, indeed reflect the NADPH redox state in intact liver cells.