Binding of the Promen fluorescent probe to human serum albumin: a fluorescence spectroscopic study.

The binding of Promen (6-propionyl-2-methoxynapthalene) to human serum albumin (HSA) was measured by fluorescence spectroscopy, finding only one class of binding sites on the protein. Hydrophobic interactions play an important role to stabilize the complex. Attempts were made to characterize its binding site using as competitors warfarin, phenylbutazone and diazepam, which bind in a specific site or region on the HSA. Fluorescence polarization measurements and spectrofluorimetric results suggest that diazepam and Promen bind at different but interacting binding sites on the HSA. The changes in the fluorescence emission of the bound Promen in the presence of these drugs, allow to use Promen to detect unspecific interactions with the site II on the HSA.     

Folding and stability of trp aporepressor from Escherichia coli

Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein. The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error. All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model. We find no evidence for stable folding intermediates. Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone. Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner. The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible. The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels.     

Binding of quercetin with human serum albumin: a critical spectroscopic study

Flavonols are plant pigments that are ubiquitous in nature. Quercetin (3,3',4',5,7-pentahydroxyflavone) and other related plant flavonols have come into recent prominence because of their usefulness as anticancer, antitumor, anti-AIDS, and other important therapeutic activities of significant potency and low systemic toxicity. Quercetin is intrinsically weakly fluorescent in aqueous solution, showing an emission maximum at approximately 538 nm. Upon binding to human serum albumin (HSA), quercetin undergoes dramatic enhancement in its fluorescence emission intensity, along with the appearance of dual emission behavior, consisting of normal and excited-state proton transfer (ESPT) fluorescence. In addition, the occurrence of a third emitting species has been noted for the first time. This is attributed to a electronic ground-state complex formed in the protein environment. High values of the fluorescence anisotropy (r) are obtained in the presence of HSA for the ESPT tautomer (r = 0.18), as well as the complex species (r = 0.37) of quercetin, indicating that the precursor ground-state molecules for both these emitting species of quercetin molecules are located in the motionally constrained sites of HSA. The steady-state emission data suggest that quercetin binds to two distinct sites in HSA from which the emissions from the normal tautomer and complex species take place. The preliminary results of studies on emission decay kinetics are also reported herein. Studies by far-UV circular dichroism spectroscopy reveal that binding of quercetin induces no significant perturbation in the secondary structure of HSA.     

Complexation of polymethine dyes with human serum albumin: a spectroscopic study

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Phif; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Phif vary from 10(4) to (5-6) x 10(7) M(-1). K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis-->trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study

Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient F?rster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.     

The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution

We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data. This is the largest protein structure thus far determined by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10. Sequence-specific 1H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332-6341]. Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach

The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT–IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were ?4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non‐radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na⁺, K⁺, Li⁺, Ni²⁺, Ca²⁺, Zn²⁺ and Al³⁺ were found to influence binding of the drug to protein. The 3D fluorescence, FT–IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular dynamics studies of a DNA-binding protein: 1. A comparison of the trp repressor and trp aporepressor aqueous simulations.

The results of two 30-ps molecular dynamics simulations of the trp repressor and trp aporepressor proteins are presented in this paper. The simulations were obtained using the AMBER molecular mechanical force field and in both simulations a 6-A shell of TIP3P waters surrounded the proteins. The trp repressor protein is a DNA-binding regulatory protein and it utilizes a helix-turn-helix (D helix-turn-E helix) motif to interact with DNA. The trp aporepressor, lacking two molecules of the L-tryptophan corepressor, cannot bind specifically to DNA. Our simulations show that the N- and C-termini and the residues in and near the helix-turn-helix motifs are the most mobile regions of the proteins, in agreement with the X-ray crystallographic studies. Our simulations also find increased mobility of the residues in the turn-D helix-turn regions of the proteins. We find the average distance separating the DNA-binding motifs to be larger in the repressor as compared to the aporepressor. In addition to examining the protein residue fluctuations and deviations with respect to X-ray structures, we have also focused on backbone dihedral angles and corepressor hydrogen-bonding patterns in this paper.     

Interaction of virstatin with human serum albumin: spectroscopic analysis and molecular modeling

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ～pH 7.2, B form ～pH 9.0 and F form ～pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants K(a) for N and B isomers were found to be 6.09×10(5 )M(-1) and 4.47×10(5) M(-1), respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1:1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.     

Interaction between hesperetin and human serum albumin revealed by spectroscopic methods

Hesperetin (5,7,3'-trihydroxyl-4'-methoxyl-flavanone) is an important bioactive compound in Chinese traditional medicine and has multiple biological and pharmacological activities. The interaction of hesperetin with human serum albumin (HSA) has been investigated by UV absorption, fluorescence and Fourier transformed infrared spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of drug at the molar ratio of drug to HSA ranging from 0.3 to 7 and the binding affinity (K(A)) was 8.11x10(4) M(-1). The primary binding site was most likely located on subdomain IIA. The binding ability of the drug to protein decreased from pH 6.4 to 8.4 in the drug to protein molar ratio of 1. Combining the curve-fitting results of infrared amide I band in D2O and H2O phosphate buffers, the alterations of protein secondary structure after drug complexation were estimated. With increasing the drug concentration, the percentage of protein alpha-helix structure decreased gradually. The reduction of protein alpha-helix structure reached about 7-9% after the protein interacted with hesperetin in D2O and H2O buffer solution at pH 7.4 when the drug to protein molar ratio was 10. This indicated a partial unfolding of HSA in the presence of the drug. From the results of UV absorption, fluorescence and Fourier transformed infrared spectrometry, the binding mode was discussed. The main mechanism of protein fluorescence quenching was a static quenching process and the hydroxyl groups of the drug in its neutral part played an important role in the binding process.     

Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli

Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that form the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of threonine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two- and four-fold, respectively. Replacement of arginine 54 by histidine (RH84) or glycine 85 by arginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants produces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimers upon treatment at 65 degrees C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of human serum albumin with 10-hydroxycamptothecin: spectroscopic and molecular modeling studies

In this work, fluorescence spectroscopy in combination with circular dichroism spectroscopy and molecular modeling was employed to investigate the binding of 10-hydroxycamptothecin (HCPT) to human serum albumin (HSA) under simulative physiological conditions. The experiment results showed that the fluorescence quenching of HSA by HCPT was a result of the formation of HCPT–HSA complex. The corresponding association constants (K _a) between HCPT and HSA at four different temperatures were determined according to the modified Stern–Volmer equation. The results of thermodynamic parameters ΔG, ΔH, and ΔS indicated that hydrogen bonds and van der Waals forces played major roles for HCPT–HSA association. Site marker competitive displacement experiment indicated that the binding of HCPT to HSA primarily took place in sub-domain IIA (site I). Molecular docking study further confirmed the binding mode and the binding site obtained by fluorescence and site marker competitive experiments. The conformational investigation showed that the presence of HCPT decreased the α-helical content of HSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of HSA molecules.     

Molecular interactions of isoxazolcurcumin with human serum albumin: spectroscopic and molecular modeling studies

Curcumin is a nontoxic natural product with diverse pharmacological potencies. We report the interaction of a potent synthetic derivative of curcumin, isoxazolcurcumin (IOC) with human serum albumin (HSA) using various biophysical methods. The observed fluorescence quenching of HSA by IOC is due to a complex formation by a static quenching process with a quenching constant of the order of 10(5) M(-1). The binding affinity and the number of binding sites were obtained from a Scatchard analysis. Thermodynamics reveals that the interaction is entropy driven with predominantly hydrophobic forces. From the observed F?rster-type fluorescence resonance energy transfer (FRET), the donor (Trp 214 in HSA) to acceptor (IOC) distance is calculated to be 3.2 nm. The conformational changes of HSA due to the interaction were investigated qualitatively from synchronous fluorescence spectra along with a quantitative estimation of the secondary structure from Fourier Transform Infrared (FTIR) and circular dichroism (CD) spectroscopies. Molecular docking studies were performed to obtain information on the possible residues involved in the interaction process, and changes in accessible surface area of the interacting residues were calculated. The preferred binding site of IOC was analyzed by ligand displacement experiments with 1-anilino-8-naphthalenesulfonate (ANS) and warfarin-bound HSA.     

Interaction of colchicine with human serum albumin investigated by spectroscopic methods

We investigated the interaction between colchicine and human serum albumin (HSA) by fluorescence and UV-vis absorption spectroscopy. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by colchicine is a result of the formation of colchicines-HSA complex; van der Waals interactions and hydrogen bonds play a major role in stabilizing the complex. The modified Stern-Volmer quenching constant K(a) and corresponding thermodynamic parameters deltaH, deltaG, deltaS at different temperatures were calculated. The distance r between donor (Trp214) and acceptor (colchicine) was obtained according to fluorescence resonance energy transfer (FRET).     

Cobinding of bilirubin and laurate to human serum albumin: spectroscopic characterization of stoichiometric complexes

Light absorption and CD spectra of bound bilirubin and albumin fluorescence spectra have been recorded from mixtures containing albumin, A, bilirubin, B, and laurate, L, in Tris-NaCl buffer at pH 8.2, 25 degrees C. Concentrations of the corresponding stoichiometric complexes, ABiLj, for i = 0/3 and j = 0/3, have been calculated from previously determined stoichiometric cobinding constants (H. Sato et al. (1988) Arch. Biochem. Biophys. 260, 811-821). Spectral data of the complexes have finally been found by iterative computer fitting using the principle of several acceptable solutions (R. Brodersen et al. (1987) Eur. J. Biochem. 169, 487-495). The results were utilized at the microscopic level to investigate ligand-induced conformational changes. When laurate was bound to AB, a decrease of the distance between Trp-214 and the bound bilirubin occurred, as measured according to F?rster's principle. The distances were 21.9 +/- 0.3 A in AB, 19.7 +/- 0.3 A in ABL, and 17.9 +/- 0.2 A in ABL2.     

Effect of guanidine hydrochloride and urea on the interaction of 6-thioguanine with human serum albumin: a spectroscopic and molecular dynamics based study

6-thioguanine (6-TG) is an antineoplastic, nucleobase guanine, purine analog drug belongs to thiopurine drug-family of antimetabolites. In the present study, we report an experimental approach towards interaction mechanism of 6-TG with human serum albumin (HSA) and examine the chemical stability of HSA in the presence of denaturants such as guanidine hydrochloride (GdnHCl) and urea. Interaction of 6-TG with HSA has been studied by various spectroscopic and spectropolarimeteric methods to investigate what short of binding occurs at physiological conditions. 6-TG binds in the hydrophobic cavity of subdomain IIA of HSA by static quenching mechanism which induces conformation alteration in the protein structure. That helpful for further study of denaturation process where change in secondary structures causes unfolding of protein that also responsible for severance of domain III from rest of the protein part. We have also performed molecular simulation and molecular docking study in the presence of denaturating agents to determine the binding property of 6-TG and the effect of denaturating agents on the structural activity of HSA. We had found that GdnHCl is more effective denaturating agent when compared to urea. Hence, this study provides straight evidence of the binding mechanism of 6-TG with HSA and the formation of intermediate or unfolding transition that causes unfolding of HSA.     

Toxic effects of chrysoidine on human serum albumin: isothermal titration calorimetry and spectroscopic investigations

Chrysoidine is widely used in industry as a type of azo dye, and is sometimes used illegally as a food additive despite its potential toxicity. Human serum albumin (HSA) is one of the most important proteins in blood plasma and possesses major physiological functions. In the present study, the conformational and functional effects of chrysoidine on HSA were investigated by isothermal titration calorimetry (ITC), multiple spectroscopic methods, a molecular docking study and an esterase activity assay. Based on the ITC results, the binding stoichiometry of chrysoidine to HSA was estimated to be 1.5:1, and was a spontaneous process via a single hydrogen bond. The binding of chrysoidine to HSA induced dynamic quenching in fluorescence, and changes in secondary structure and in the microenvironment of the Trp‐214 residue. In addition, the hydrogen bond (1.80 Å) formed between the chrysoidine molecule and the Gln‐211 residue. The esterase activity of HSA decreased following the addition chrysoidine due to the change in protein structure. This study details the direct interaction between chrysoidine and HSA at the molecular level and the mechanism for toxicity as a result of the functional changes induced by HSA structural variation upon binding to chrysoidine in vitro. This study provides useful information towards detailing the transportation mechanism and toxicity of chrysoidine in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan

We have examined the interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan, using both equilibrium dialysis and flow dialysis methods. Results obtained by the two procedures were equivalent and indicate that the trp aporepressor binds L-tryptophan with an equilibrium dissociation constant (Kd) of 40 microM at 25 degrees C under standard binding assay conditions (10 mM potassium phosphate, pH 7.4, 0.2 M potassium chloride, 0.1 mM EDTA, 5% glycerol). Molecular sizing of the purified trp aporepressor shows that in the absence of ligand the regulatory protein exists as a dimeric species with greater than 99% purity and an apparent molecular weight of 30,000. Under the storage and assay conditions used, the dimer appears quite stable, and essentially no monomer or higher multimeric species are detected. Analysis of binding data by Scatchard and direct linear plot methods shows two identical and independent ligand-binding sites/native trp aporepressor dimer. When examined as a function of temperature, L-tryptophan binding by trp aporepressor varied over 7-fold (Kd = 28 microM at 6.5 degrees C to Kd = 217 microM at 40 degrees C). At the optimal growth temperature for E. coli (37 degrees C), the dissociation constant was 160 microM for the ligand, L-tryptophan. From the relationship between temperature and L-tryptophan binding by trp aporepressor, the apparent enthalpy change delta H = -10.6 +/- 0.6 kcal mol-1 and the apparent entropy change delta S = -17 +/- 2 cal degree-1 mol-1 were determined.