A Fluorescence Analysis of ANS Bound to Bovine Serum Albumin: Binding Properties Revisited by Using Energy Transfer

Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman-Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job's plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites (>10(4) M(-1)) are located at a reasonably close distance (18-27 A) to at least one of two tryptophan residues, while the lowest affinity binding site (approximately 10(4) M(-1)) is located over 34 A away from the both tryptophans.     

Structure of molecular associates of fluorescent probes in solutions of human serum albumin

Spectral-luminescent characteristics and molecular association processes in solutions of human serum albumin are analyzed at different pH values for three fluorescent probes (eosin, erythrosin, and fluorescein). Common features for all three probes in protein solutions are quenching of the fluorescence, a red shift of the fluorescence maximum, a decrease in the degree of association, and an increase in the angle between dipole moments of dye molecules in dimers. This being so, differences between fluorescein and its halogen derivatives (eosin and erythrosin) are observed in the pH dependences of fluorescence, degree of association, and the angle between dipole moments of probe molecules in dimers. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 6, pp. 782–787, November–December, 2008.     

Interaction of molecules of the neonicotinoid imidacloprid and its structural analogs with human serum albumin

We have used fluorescence spectroscopy methods to show that imidacloprid and its structural analogs form complexes with human serum albumin (HSA). The nature of the spectral changes in the ligand×protein systems and the calculated complexation parameters suggest that these low molecular weight compounds mainly bind to a specific section of the protein molecule, near the tryptophan residue in the 214 position of the polypeptide chain. We have found that the association constants are on the order of 10⁴ M⁻¹, and the affinity of the ligands for HSA varies in the series 6-chloronicotinic acid > 6-methoxynicotinic acid = imidacloprid > the keto analog of imidacloprid. The major contribution to the complexation energy probably comes from hydrophobic interaction forces with participation of the aromatic pyridine ring of the ligands, while additional enhancement of ligand-protein affinity can be provided by the nitroimine group of imidacloprid. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 6, pp. 859–866, November–December, 2008.     

Luminescent spectral properties of rhodamine derivatives while binding to serum albumin

We have studied the effect of blood serum albumin on the absorption and fluorescence spectra of rhodamine C (RC), rhodamine 6G (R6G), and rhodamine 3B (R3B). Interaction of the dye with protein is assessed using the binding parameters: binding constants and concentrations of binding sites. We have studied the effect of temperature on the binding parameters. We have observed that heating a mixture of the dye solution with protein for 30 min leads to an increase in the binding constant for rhodamine 3B with protein by a factor of 2, while the concentration of binding sites increases by a factor of 2.3. This is explained by features of the globular protein structure and a change in its conformation when heated. We have shown that rhodamine 3B at a concentration of 10⁻⁵ M is the most effective among the studied rhodamine dyes for application as a fluorescent probe when studying conformational changes in blood serum protein. Report given at the Third International Conference on Liquid State Physics: Current Problems, May 27–31, 2005, Kiev, Ukraine. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 3, pp. 380–384, May–June, 2006.     

Determining the binding affinity and binding site of bensulfuron-methyl to human serum albumin by quenching of the intrinsic tryptophan fluorescence

Bensulfuron-methyl (BM) is a highly active sulfonylurea herbicide for use on paddy rice. Steady state fluorescence, UV/vis absorption, circular dichroism (CD), time-resolved fluorescence and molecular modeling methods have been exploited to determine the binding affinity and binding site of BM to human serum albumin (HSA). From the synchronous fluorescence, UV/vis, CD and three-dimensional fluorescence spectra, it was evident that the interaction between BM and HSA induced a conformational change in the protein. Steady state and time-resolved fluorescence data illustrates that the fluorescence quenching of HSA by BM was the formation of HSA-BM complex at 1:1 molar ratio. Site marker competitive experiments demonstrated that the binding of BM to HSA primarily took place in subdomain IIIA (Sudlow’s site II), this corroborates the hydrophobic probe ANS displacement and molecular modeling results. Thermodynamic analysis displays hydrophobic, electrostatic and hydrogen bonds interactions are the major acting forces in stabilizing the HSA-BM complex.     

Fluorescence lifetime-enhanced spectral characterization of human serum

Frequency-domain fluorescence lifetime techniques were used for the characterization of pooled human serum, including normal serum, hyperlipid serum, and sera that had been stripped of various components. Fluorescence lifetime measurements of normal human serum revealed lifetime components primarily in the regions of 10² ps, 1–2 ns, 4–7 ns, and 9–10 ns. Phase-resolved fluorescence spectroscopy (PRFS), a frequency-domain technique that combines spectral and lifetime information, in measurements of phase-resolved fluorescence intensity (PRFI), provided the basis for comparison of the various sera. Measurements of PRFI vs excitation wavelength and emission wavelength yield a phase-resolved excitation-emission matrix (PREEM) at a given modulation frequency. Multifrequency measurements yield a three-way excitation-emission-frequency array. The multifrequency PREEMs of the various sera were compared with each other and with the corresponding two-way excitation-emission matrices (EEMs) that are obtained using conventional, steady-state fluorescence spectroscopy. Application of matrix-based analysis techniques to the steady-state and PRFS data arrays allowed direct comparison between the two approaches. Results demonstrate the enhanced discrimination among samples that is achieved through the additional dimension of fluorescence lifetime in PRFS.     

Fluorescence spectrometric studies on the binding of puerarin to human serum albumin using warfarin, ibuprofen and digitoxin as site markers with the aid of chemometrics

The interaction of puerarin with human serum albumin (HSA) in pH 7.4 Tris-HCl buffer has been investigated by fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. The results revealed the presence of static type of quenching mechanism in the binding of puerarin to HSA. The association constants (K_a) between puerarin and HSA were obtained according to Modified Stern-Volmer equation. The calculated thermodynamic parameters indicated that the binding of puerarin to HSA was driven mainly by hydrophobic interaction. The competitive experiments of site markers suggested that the binding site of puerarin to HSA was located in the region of subdomain IIA (sudlow site I). Further, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way synchronous fluorescence spectra data of the competitive interaction between puerarin and warfarin with HSA. The concentration information for the three reaction components, warfarin, puerarin and puerarin−HSA, in the system at equilibrium was obtained simultaneously. The PARAFAC analysis indicated that puerarin in the puerarin-HSA complex was displaced by warfarin, which confirmed the binding site of puerarin to HSA was located in site I. Moreover, the results of CD and FT-IR spectra demonstrated that the secondary structure of HSA was changed in the presence of puerarin.     

Complexation of fluoroquinolone antibiotics with human serum albumin: A fluorescence quenching study

Mechanism of interaction and detailed physico-chemical characterization of the binding of four fluoroquinolones: levofloxacin, sparfloxacin, ciprofloxacin HCl and enrofloxacin with human serum albumin has been studied at physiological pH (7.4) using fluorescence spectroscopic technique. The stoichiometry of interaction was found to be 1:1 for all the drugs used. The association constants for the interaction were of the order of 10⁴ in most cases. At low drug:protein ratios, a significant fraction of the added drug was bound. The predominant interactions involved are hydrogen bonding and Van der Waal’s interactions in the case of levofloxacin, hydrophobic interactions in the case of ciprofloxacin hydrochloride and enrofloxacin and hydrogen bonding, hydrophobic and electrostatic interactions in the case of sparfloxacin.The drug binding region did not coincide with that of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS). From the displacement of site-specific probes and site-marker drugs, it was concluded that ciprofloxacin hydrochloride is site II-specific while enrofloxacin is a site I-specific drug. Levofloxacin binds at both site I and site II with equal affinity. Sparfloxacin had higher affinity for site II than site I. It is also possible that sparfloxacin binds at the interface between site I and site II. Stern-Volmer analysis of the data showed that the quenching mechanism is predominantly collisional for the binding of ciprofloxacin HCl and enrofloxacin while both static and collisional quenching mechanisms are operative in the case of levofloxacin and sparfloxacin. High magnitude of the rate constant for quenching showed that the process is not entirely diffusion controlled. Circular dichroism (CD) spectroscopic studies showed that the presence of drugs did not cause any major changes in the secondary structure of HSA.     

Probing the binding of vitexin to human serum albumin by multispectroscopic techniques

The interaction between vitexin and human serum albumin (HSA) has been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of vitexin to HSA. The binding constants (K_a) between vitexin and HSA were obtained according to the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -57.29 kJ mol⁻¹ and -99.01 J mol⁻¹ K⁻¹ via the van't Hoff equation, which indicated that the interaction of vitexin with HSA was driven mainly by hydrogen bond and van der Waals forces. Fluorescence anisotropy data showed that warfarin and vitexin shared a common binding site I corresponding to the subdomain IIA of HSA. The binding distance (r) between the donor (HSA) and the acceptor (vitexin) was 4.16 nm based on the Förster theory of non-radioactive energy transfer. In addition, the results of synchronous fluorescence, CD and FT-IR spectra demonstrated that the microenvironment and the secondary structure of HSA were changed in the presence of vitexin.     

Time-Resolved Fluorescence Studies on Bovine Serum Albumin Denaturation Process

The denaturation of Bovine Serum Albumin (BSA) by a chaotropic agent, guanidinium hydrochloride (GuH+Cl-) was studied by fluorescence lifetime analysis. The BSA was labelled with 1-anilino-8-naphthalene sulfonate (ANS) at two different molar ratios (1:1) and (1:10). The non-exponential fluorescence kinetics of the BSA-ANS complex at different stages of denaturation is analysed using three different models: a discrete tri-exponential sum, stretched exponential, and Gaussian lifetime distribution. In all cases, the fluorescence decay times decreased with protein denaturation. The results from the models show that there are at least two different binding sites located in the BSA protein with different water accessibility.     

三种异黄酮类药物与不同异构体人血清白蛋白的作用机制研究

利用荧光光谱研究了三种异黄酮类化合物染料木素、鸡豆黄素A和3’,4’,7-三羟基异黄酮与不同异构体人血清白蛋白的相互作用机制。计算了异黄酮与蛋白质形成复合物的各种结合参数(猝灭速率常数、结合常数及结合位点数),结果表明三种异黄酮类化合物在人血清白蛋白上只有一个结合位点,位于结合位点siteⅠ,结合常数在0.17×105～1.20×105 L.mol-1之间。荧光增强光谱结果显示,与蛋白质作用后药物的荧光强度明显增加,说明了药物与人血清白蛋白发生了结合,在此基础上讨论了三种异黄酮与人血清白蛋白的结合机理。     

Molecular spectroscopic insight into the binding of batatasin V isomers to human serum albumin

Fluorescence, ultraviolet-visible absorption spectroscopy, circular dichroism spectrum, and molecular docking methods were employed to study the interaction mechanisms of batatasin V and its isomer with human serum albumin. The two isomers both bond reactively to the hydrophobic activity in subdomain IIA, with an approximate binding affinity. Thermodynamic parameters and molecular modeling results manifested that hydrogen bonds and van der Waals force were the main contributors to the interaction. The secondary structure of human serum albumin was altered with the obvious decreased amount of α-helix. The results overall suggested similar binding mechanisms of batatasin V isomers with human serum albumin. This work will promote the further study of batatasins for pharmacological function. It could also help to provide some useful information for further drug design based on batatasins.     

Insights into the ninhydrin chemiluminescent reaction and its potential for micromolar determination of human serum albumin

The N-terminal region of human serum albumin (HSA) has an inherent affinity for Co(II) ions. On this basis a new continuous flow method for detection of HSA has been developed taking advantage of the strong quenching effect of the albumin in the ninhydrin-H₂O₂-Co(II) chemiluminescent system. The analytical potential of the system is compared with other conventional chemiluminescent reagents. The method gives linear responses from the detection limit (0.30 μM HSA) up to 6.8 μM. The repeatability of the method is good (RSD=7%), it is cheap and rapid to apply and does not require the use of insoluble or expensive reagents nor sophisticated equipment.     

Spectroscopic determination of binding between human serum albumin and a platinum(II) dimethylsulfoxide complex

We have used electronic absorption and fluorescence spectroscopy to study binding between a platinum(II) dimethylsulfoxide complex (cis-[Pt(DMSO)₂Cl₂]) and human serum albumin (HSA), and the effect of complexation on the structure of the protein. We have calculated the binding parameters for binding between cis-[Pt(DMSO)₂Cl₂] and HSA. We have determined the binding constant K_B = (1.2 ± 0.1)·10³ M⁻¹ and the Hill coefficient h = 1.03 ± 0.1. We have determined that binding between cis-[Pt(DMSO)₂Cl₂] and the protein leads to a change in the internal packing of the macromolecule. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 4, pp. 573–576, July–August, 2008.     

Interaction of the docetaxel with human serum albumin using optical spectroscopy methods

Docetaxel is a semi-synthetic product derived from the needles of the European yew. It is an antineoplastic agent belonging to the taxoid family. The interaction between docetaxel and human serum albumin (HSA) has been investigated systematically by the fluorescence quenching technique, synchronous fluorescence spectroscopy, ultraviolet (UV)-vis absorption spectroscopy, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) under physiological conditions. Our fluorescence data showed that HSA had only one docetaxel binding site and the binding process was a static quenching procedure. According to the Van’t Hoff equation, the thermodynamic parameters standard enthalpy (ΔH⁰) and standard entropy (ΔS⁰) were calculated to be −41.07 KJ mol⁻¹ and −49.72 J mol⁻¹ K⁻¹. These results suggested that hydrogen bond was the predominant intermolecular force stabling the docetaxel-HSA complex. The data from the CD, FT-IR and UV-vis spectroscopy supported the change in the secondary structure of protein caused by the interaction of docetaxel with HSA.     

Complexes between C.I. Acid Orange 6 and human serum albumin, a multi-spectroscopic approach to investigate the binding behavior

The interaction mechanism of Acid Orange 6 (AO6) with human serum albumin (HSA) was investigated firstly by using fluorescence quenching technique, UV absorbance, circular dichroism (CD), Fourier transform infrared (FT-IR), three-dimensional fluorescence spectroscopy in combination with molecular modeling method under simulative physiological conditions. Fluorescence data indicated that there is a single class of binding sites between AO6 and HSA, and the alterations of HSA secondary structure in the presence of AO6 was confirmed by synchronous fluorescence, UV, CD, FT-IR and three-dimensional fluorescence spectra. The efficiency of fluorescence resonance energy transfer provided the binding distance (r) of 2.83 nm for AO6-HSA system. Furthermore, the thermodynamic parameters enthalpy change (ΔH⁰) and entropy change (ΔS⁰) for the reaction were calculated to be −5.77 kJ mol⁻¹ and 109.42 J mol⁻¹ K⁻¹, respectively, according to Van't Hoff equation, these data suggested that both hydrophobic forces and hydrogen bonding play a major role in the binding of AO6 to HSA, which agrees well with the results of molecular modeling study. Experimental results showed that the interaction between AO6 and HSA induced a conformational change of HSA, which was proved by the qualitative and quantitative analysis data of different spectroscopic techniques under simulative physiological conditions.     

Luminescent spectral characteristics of eosin in solutions of human serum albumin when denatured by treatment with sodium dodecyl sulfate

From analysis of the fluorescence spectra of eosin molecules in a solution with human serum albumin (HSA), we have obtained information about the dynamics of protein conformational rearrangements during denaturing of the protein when treated with sodium dodecyl sulfate (SDS) for different pH values of the solution. We hypothesize that HSA denaturing in the presence of SDS occurs in two stages: the first stage is loosening of the protein globules, and the second stage is complete unfolding of the protein molecules. We have shown that denaturating of the protein in the presence of SDS passes through both stages for a solution pH below the isoelectric point of the albumin, while the denaturing stops in the first stage for a solution pH above the isoelectric point of the albumin. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 5, pp. 661–665, September–October, 2006.     

Extrinsic fluorescence probe study of human serum albumin using Nile red

Nile red bound to human serum albumin (HSA) shows an order of magnitude increase in the probe's fluorescence intensity. Here, we report on the fluorescence characteristics of the probe-protein complex in Trizma buffer (pH 7.1), urea, guanidine hydrochloride, and AOT/isooctane/buffer reverse micelles using both steady—state and time-resolved fluorescence techniques. With a view to illustrating the use of extrinsic probe fluorescence spectroscopy in protein research, we demonstrate that protein unfolding can be observed through measurements of the probe's time-resolved anisotropy and steady-state fluorescence spectrum. Moreover, this shows that thermal unfolding is fundamentally different from using denaturant, with respect to changes in both the nanosecond diffusional rotation of the probe at intermediate stages and in the denatured protein's structure. Also, the large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size to be easily identified in the steady-state fluorescence. This was not seen in earlier work exploiting the intrinsic tryptophan fluorescence of HSA and further demonstrates the complementary information that extrinsic fluorescence probe studies can offer protein science. We discuss the complex acrylamide quenching characteristics of Nile red bound to HSA in terms of the possibility of at least two binding sites for the probe and the effect of acrylamide on the probe-protein structure at very high quencher concentrations.     

Complexation of insecticide chlorantraniliprole with human serum albumin: Biophysical aspects

Chlorantraniliprole is a novel insecticide belonging to the diamide class of selective ryanodine receptor agonists. A biophysical study on the binding interaction of a novel diamide insecticide, chlorantraniliprole, with staple in vivo transporter, human serum albumin (HSA) has been investigated utilizing a combination of steady-state and time-resolved fluorescence, circular dichroism (CD), and molecular modeling methods. The interaction of chlorantraniliprole with HSA gives rise to fluorescence quenching through static mechanism, this corroborates the fluorescence lifetime outcomes that the ground state complex formation and the predominant forces in the HSA-chlorantraniliprole conjugate are van der Waals forces and hydrogen bonds, as derived from thermodynamic analysis. The definite binding site of chlorantraniliprole in HSA has been identified from the denaturation of protein, competitive ligand binding, and molecular modeling, subdomain IIIA (Sudlow's site II) was designated to possess high-affinity binding site for chlorantraniliprole. Moreover, using synchronous fluorescence, CD, and three-dimensional fluorescence we testified some degree of HSA structure unfolding upon chlorantraniliprole binding.     

Characterization of sonochemically prepared human serum albumin nanocapsules using different plant oils as core component for targeted drug delivery

The focus of this study is the preparation of proteinaceous human serum albumin (HSA) nanocapsules with biocompatible plant oil cores avoiding toxic cross-linker and noxious non-aqueous liquids. The sonochemical preparation of HSA capsules with different plant oils yields particles with narrow size distribution forming suspensions stable for at least 14 days and enabling long-term storage by freezing. Furthermore, wheat germ agglutinin (WGA) as a targeting molecule was successfully embedded into the proteinaceous particle shell at a molar ratio of 7:1 (HSA/WGA). As urothelial cell binding studies revealed up to 55% higher cell binding potential of WGA-grafted particles than those without a targeter, targeted protein nanocapsules represent the first step towards new and innovative formulations.