Association of hemin with protein 4.1 as compared to spectrin and actin

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 . 10(7) M-1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.     

A novel amphiphilic thiosemicarbazone derivative for binding and selective sensing of human serum albumin

The interaction of ligands and drug molecules with protein is of major interest in drug pharmacokinetics and pharmacodynamics. In this study, we synthesized a novel thiosemicarbazone‐based amphiphilic molecule for selective binding and detection of human serum albumin (HSA) with significant increase in fluorescence intensity. The compound 5‐(octyloxy) naphthalene substituted salicylaldehyde thiosemicarbazone was designed to interact with site I of HSA. The weak fluorescence of the probes in aqueous solution showed a dramatic increase in fluorescence intensity upon binding with HSA, while the responses to various other proteins and enzymes were negligible under similar experimental conditions. Changes in fluorescence intensity and formation of a new emission maximum of the compound in the presence of HSA as well as an increase in steady‐state anisotropy values reflected well the nature of binding and location of the probe inside the protein environment. Copyright © 2012 John Wiley & Sons, Ltd.     

Drug binding to human serum albumin: abridged review of results obtained with high-performance liquid chromatography and circular dichroism

The drug binding to plasma and tissue proteins are fundamental factors in determining the overall pharmacological activity of a drug. Human serum albumin (HSA), together with alpha1-acid glycoprotein (AGP), are the most important plasma proteins, which act as drug carriers, with drug pharmacokinetic implications, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This review focuses on the combination of biochromatography and circular dichroism as an effective approach for the characterization of albumin binding sites and their enantioselectivity. Furthermore, their applications to the study of changes in the binding properties of the protein arising by the reversible or covalent binding of drugs are discussed, and examples of physiological relevance reported. Perspectives of these studies reside in supporting the development of new drugs, which require miniaturization to facilitate the screening of classes of compounds for their binding to the target protein, and a deeper characterization of the mechanisms involved in the molecular recognition processes.     

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples.     

The disaccharide anthracycline MEN 10755 binds human serum albumin to a non-classical drug binding site

The interaction of the novel disaccharide anthracycline MEN 10755 with human serum albumin (HSA) was investigated by visible absorption and fluorescence spectroscopies and by ultrafiltration. Notably, MEN 10755 binds serum albumin far stronger than doxorubicin. Albumin binding results into a drastic quenching of the intrinsic fluorescence of MEN 10755; a binding constant of 1.1 x 10(5) was determined from fluorescence data. To localize the HSA binding site of MEN 10755 competition experiments were carried out with ligands that are selective for the different drug binding sites of the protein. No relevant competition effects were seen in the case of warfarin, diazepam and hemin, known ligands of sites I, II and III, respectively. Modest effects were observed following addition of palmitic acid that targets the several fatty acid binding sites of the protein. In contrast, extensive displacement of the bound anthracycline was achieved upon addition of ethacrinic acid. On the basis of these results, it is proposed that MEN 10755 binds serum albumin tightly to a non-canonical surface binding site for which it competes specifically with ethacrinic acid.     

Participation of the lone tryptophan residue of rat alpha-foetoprotein in its drug-binding sites. Comparison with rat serum albumin.

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.     

Spectroscopic and in silico study of binding mechanism of cynidine‐3‐O‐glucoside with human serum albumin and glycated human serum albumin

The drug–serum albumin interaction plays a dominant role in drug efficacy and disposition. The glycation of serum albumin that occurs during diabetes may affect its drug‐binding properties in vivo. In order to evaluate the interactivity characteristics of cyanidin‐3‐O‐glucoside (C3G) with human serum albumin (HSA) and glycated human serum albumin (gHSA), this study was undertaken using multiple spectroscopic techniques and molecular modeling analysis. Time‐resolved fluorescence and the thermodynamic parameters indicated that the quenching mechanism was static quenching, and hydrogen bonding and Van der Waals force were the main forces. The protein fluorescence could be quenched by C3G, whereas the polarity of the fluorophore was not obviously changed. C3G significantly altered the secondary structure of the proteins. Furthermore, the interaction force that existed in the HSA–C3G system was greater than that in the gHSA–C3G system. Fluorescence excitation emission matrix spectra, red edge excitation shift, Fourier transform infrared spectroscopy and circular dichroism spectra provided further evidence that glycation could inhibit the binding between C3G and proteins. In addition, molecular modeling analysis supported the experimental results. The results provided more details for the application of C3G in the treatment of diabetes.     

Study of the interaction of 2'-5'-oligoadenylates and their analogues with proteins by fluorescence spectroscopy

The interaction of "core" 2'-5'-oligoadenylates (2-5A) and their analogues with proteins albumin, interferon and immunoglobulin G was studied by fluorescence spectroscopy methods. Strong quenching of protein fluorescence (up to 67%) was observed upon interaction of oligoadenylates in concentration of 5 x 10(-5) M with albumin. Investigated compounds quenched the emission of interferon to a lesser extent, whereas no significant fluorescence changes occurred upon interaction with immunoglobulin under the same conditions. The level of quenching depended on the structure of 2-5A compounds and decreased with the decrease of their concentration. These data suggest that 2-5A actively bind to albumin and less efficiently to interferon, and practically do not interact with immunoglobulin. Taking into consideration different efficiency of quenching of the fluorescence excited at 280 and 296 nm, the assumption has been made about a possible role of tyrosine and tryptophan in the binding of a given compound to proteins. Possible mechanisms of interaction of oligoadenylates with proteins are discussed.     

Symmetric and asymmetric squarylium dyes as noncovalent protein labels: a study by fluorimetry and capillary electrophoresis

Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), beta-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10(5) and 10(7) were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.     

Influence of quercetin on the interaction of gliclazide with human serum albumin – spectroscopic and docking approaches

Protein‐binding interactions are displacement reactions which have been implicated as the causative mechanisms in many drug–drug interactions. Thus, the aim of presented study was to analyse human serum albumin‐binding displacement interaction between two ligands, hypoglycaemic drug gliclazide and widely distributed plant flavonoid quercetin. Fluorescence analysis was used in order to investigate the effect of substances on intrinsic fluorescence of human serum albumin (HSA) and to define binding and quenching properties of ligand–albumin complexes in binary and ternary systems, respectively. Both ligands showed the ability to bind to HSA, although to a different extent. The displacement effect of one ligand from HSA by the other one has been described on the basis of the quenching curves and binding constants comparison for the binary and ternary systems. According to the fluorescence data analysis, gliclazide presents a substance with a lower binding capacity towards HSA compared with quercetin. Results also showed that the presence of quercetin hindered the interaction between HSA and gliclazide, as the binding constant for gliclazide in the ternary system was remarkably lower compared with the binary system. This finding indicates a possibility for an increase in the non‐bound fraction of gliclazide which can lead to its more significant hypoglycaemic effect. Additionally, secondary and tertiary structure conformational alterations of HSA upon binding of both ligands were investigated using synchronous fluorescence, circular dichroism and FT‐IR. Experimental data were complemented with molecular docking studies. Obtained results provide beneficial information about possible interference upon simultaneous co‐administration of the food/dietary supplement and drug.     

The interaction of blood proteins with brucine

The features of brucine (BC) binding to two blood proteins, bovine hemoglobin (BHb), and bovine serum albumin (BSA), were investigated via fluorescence, circular dichroism and UV/Vis absorption spectroscopy. The results revealed that BC caused the fluorescence quenching of blood proteins by the formation of BC–protein complex. The corresponding thermodynamic parameters were measured at different temperatures. The process of binding BC molecule on protein was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The molecular docking has been employed to explore the binding site of the BC in BHb and BSA on the Autodock 4.2. The distances r between BC and protein were calculated to be 4.93 and 5.08 nm for BHb, and BSA, respectively. The effect of BC on the conformation of blood proteins was analyzed using CD, synchronous fluorescence and three-dimensional fluorescence spectra.     

A new and simple procedure for the evaluation of the association of surfactants to proteins

A new and simple method useful for the evaluation of the association of surfactants to proteins is proposed. The method is based on an analysis of the effect promoted by surfactant addition upon the fluorescence intensity of the intrinsic tryptophan chromophore and its dependence with protein concentration. The proposed methodology is applied to quantify the binding of an anionic (sodium dodecylsulfate), a zwitterionic (N-hexadecyl-N,N′-dimethyl-3-ammonio-1-propane-sulfonate) and a neutral (Triton X-100, reduced) surfactant to bovine serum albumin (BSA).     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.     

Complexation of beta-lactam antibiotic drug carbenicillin to bovine serum albumin: energetics and conformational studies

Binding of the antibiotic drug carbenicillin to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism (CD) spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, and in the presence of anionic, cationic and nonionic surfactants, tetrabutylammonium bromide, and sucrose. The values of van't Hoff enthalpy do not agree with the calorimetric enthalpy indicating conformational changes in the protein upon drug binding. These observations are supported by the intrinsic fluorescence and CD spectroscopic measurements. A reduction in the binding affinity of carbenicillin to BSA is observed with increase in ionic strength of the solution, thereby suggesting, prevailing of electrostatic interactions in the binding process. The involvement of hydrophobic interactions in the binding of the drug to the protein is also indicated by a slight reduction in binding constant in the presence of tetrabutylammonium bromide. The experiments in the presence of sucrose suggest that hydrogen bonding is perhaps not dominant in the binding. The anionic surfactant sodium dodecyl sulphate (SDS) is observed to completely interfere in the ionic interactions in addition to its partial denaturing capacity. However, the presence of cationic surfactant hexadecyl trimethylammonium bromide (HTAB) and nonionic surfactant Triton-X 100 induce a slight reduction in the values of binding affinity. These calorimetric and spectroscopic results, provide quantitative information on the binding of carbenicillin to BSA and suggests that the binding is dominated by electrostatic interactions with contribution from hydrophobic interactions. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 831-840, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Investigation on the effect of fluorescence quenching of bovine serum albumin by cefoxitin sodium using fluorescence spectroscopy and synchronous fluorescence spectroscopy

The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of the synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanisms of drugs with protein and was consistent with one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between drug and protein and energy‐transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role in the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. The primary binding site for cefoxitin sodium was sub‐hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill's coefficients (n_H) was approximately equal to 1, which suggested no cooperativity in the bovine serum albumin–cefoxitin sodium system. The donor‐to‐acceptor distance r < 7 nm indicated that static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non‐radiation energy‐transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative studies on the interaction of cefixime with bovine serum albumin by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy

Under simulated physiological conditions, the reaction mechanism between cefixime and bovine serum albumin at different temperatures (293, 303 and 310 K) was investigated using a fluorescence quenching method and synchronous fluorescence method, respectively. The results indicated that the fluorescence intensity and synchronous fluorescence intensity of bovine serum albumin decreased regularly on the addition of cefixime. In addition, the quenching mechanism, binding constants, number of binding sites, type of interaction force and energy‐transfer parameters of cefixime with bovine serum albumin obtained from two methods using the same equation were consistent. The results indicated that the synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2014 John Wiley & Sons, Ltd.     

Magnetic core/shell Fe3O4/Au nanoparticles for studies of quinolones binding to protein by fluorescence spectroscopy

Magnetic core/shell Fe₃O₄/Au nanoparticles were used in the determination of drug binding to bovine serum albumin (BSA) using a fluorescence spectroscopic method. The binding constants and number of binding sites for protein with drugs were calculated using the Scatchard equation. Because of their superparamagnetic and biocompatible characteristics, magnetic core/shell Fe₃O₄/Au nanoparticles served as carrier proteins for fixing proteins. After binding of the protein to a drug, the magnetic core/shell Fe₃O₄/Au nanoparticles–protein–drug complex was separated from the free drug using an applied magnetic field. The free drug concentration was obtained directly by fluorescence spectrometry and the proteins did not influence the drug determination. So, the achieved number of binding sites should be reliable. The binding constant and site number for ciprofloxacin (CPFX) binding to BSA were 2.055 × 10⁵ L/mol and 31.7, and the corresponding values for norfloxacin (NOR) binding to BSA were 1.383 × 10⁵ L/mol and 38.8. Based on the achieved results, a suitable method was proposed for the determination of binding constants and the site number for molecular interactions. The method was especially suitable for studies on the interactions of serum albumin with the active ingredients of Chinese medicine. Copyright © 2015 John Wiley & Sons, Ltd.     

Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool: Nanomolar human serum albumin quantitation

The commercially available dye, NanoOrange, has been investigated as a potential tool for clinical diagnostics due to its low cost, ease of use, and ability to detect nanomolar concentrations of protein. Virtually non-fluorescent in dilute aqueous solutions, NanoOrange fluorescence is enhanced by at least an order of magnitude upon non-covalent interaction with proteins. These features, coupled with the requirement for high throughput assays in the clinical laboratory has prompted the development of two orthogonal NanoOrange approaches. Human serum albumin (HSA) was used as a model protein for the development of both 96-well microplate and capillary electrophoresis laser-induced fluorescence (CE–LIF) assay formats. Dye performance in five commonly used buffers of various concentrations and pH indicated considerable flexibility in assay buffer selection, with optimal performance at pH 9.0. A salt concentration study indicated that increasing NaCl concentration generally decreases fluorescence emission and can be minimized by pre-diluting biological samples to a final salt concentration of 20–80 mM. Titration of protein with NanoOrange resulted in optimal HSA–NanoOrange complex formation utilizing 1× and 2× NanoOrange in the 96-well microplate and CE–LIF approaches, respectively. A NanoOrange binding model based on rapid signal enhancement and zero order fluorescence emission kinetics is proposed. The utilization of NanoOrange in CE–LIF based human serum analysis results in a signal-to-background ratio improvement of up to two orders of magnitude.