Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin

Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB) > multi-metal binding site (MBS) > N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9 ± 5, 1.1 ± 0.5, and 0.9 ± 0.3 × 10⁴ M⁻¹, respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.     

Determining the binding site and binding affinity of estradiol to human serum albumin and holo-transferrin: fluorescence spectroscopic,isothermal titration calorimetry and molecular modeling approaches

The interactions between estradiol and two carrier proteins, i.e. human serum albumin (HSA) and holo-transferrin (HTF) in aqueous solution at pH = 7.4 were studied by three-dimensional fluorescence emission spectroscopy, isothermal titration calorimetry (ITC), zeta-potential, resonance light-scattering and molecular modeling. Extensive fluorescence quenching was observed throughout the interaction between the drug and both proteins. Moreover, conformational changes were determined by observing the rearrangement of Trp residues during binding of estradiol with HSA and HTF at different concentrations. ITC experiments revealed that, in the presence of estradiol, both van der Waals forces and hydrogen bonding became predominant. In addition, other binding parameters such as enthalpy and entropy changes were determined by the zeta potential method. Molecular modeling suggested that estradiol was situated within sub-domain IB sited in the hydrophobic cluster in Site I, whereas the drug was located in the N-terminal of HTF where it was hydrogen bonded with Ala 670.     

A combined spectroscopic and molecular docking approach to characterize binding interaction of megestrol acetate with bovine serum albumin

The binding interactions between megestrol acetate (MA) and bovine serum albumin (BSA) under simulated physiological conditions (pH 7.4) were investigated by fluorescence spectroscopy, circular dichroism and molecular modeling. The results revealed that the intrinsic fluorescence of BSA was quenched by MA due to formation of the MA–BSA complex, which was rationalized in terms of a static quenching procedure. The binding constant (K_b) and number of binding sites (n) for MA binding to BSA were 2.8 × 10⁵ L/mol at 310 K and about 1 respectively. However, the binding of MA with BSA was a spontaneous process due to the negative ∆G⁰ in the binding process. The enthalpy change (∆H⁰) and entropy change (∆S⁰) were – 124.0 kJ/mol and –295.6 J/mol per K, respectively, indicating that the major interaction forces in the binding process of MA with BSA were van der Waals forces and hydrogen bonding. Based on the results of spectroscopic and molecular docking experiments, it can be deduced that MA inserts into the hydrophobic pocket located in subdomain IIIA (site II) of BSA. The binding of MA to BSA leads to a slight change in conformation of BSA but the BSA retained its secondary structure, while conformation of the MA has significant change after forming MA–BSA complex, suggesting that flexibility of the MA molecule supports the binding interaction of BSA with MA. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Flavonoid binding to human serum albumin

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3 × 10⁻⁶ and 3.9 × 10⁻⁵ M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.     

Combined multispectroscopic and molecular docking investigation on the interaction between strictosamide and human serum albumin

The interaction between strictosamide (STM) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling under physiological pH 7.4. STM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding site number n and apparent binding constant K_a were determined at different temperatures by fluorescence quenching. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated as ?3.01 kJ/mol and 77.75 J/mol per K, respectively, which suggested that the hydrophobic force played major roles in stabilizing the HSA–STM complex. The distance r between donor and acceptor was obtained to be 4.10 nm according to Förster's theory. After the addition of STM, the synchronous fluorescence and three‐dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the circular dichroism spectral results showed that the α‐helix content of HSA decreased (from 61.48% to 57.73%). These revealed that the microenvironment and conformation of HSA were changed in the binding reaction. Furthermore, the study of molecular modeling indicated that STM could bind to site I of HSA and the hydrophobic interaction was the major acting force, which was in agreement with the binding mode study. Copyright © 2013 John Wiley & Sons, Ltd.     

The effect of non-enzymatic glycation on the unfolding of human serum albumin

We monitored the unfolding of human serum albumin (HSA) and glycated human serum albumin (gHSA) subjected to guanidine hydrochloride (GndHCl) by using fluorescence and circular dichroism (CD) spectroscopy. A two-state model with sloping baselines best described the Trp-214 fluorescence unfolding measurements, while a three-state model best described the far-UV CD unfolding data. Glycation of HSA increased the [D](50%) point by approximately 0.20M. This corresponded to an increase in the free energy of unfolding of gHSA relative to HSA of 2.6kJ/mol. The intrinsic fluorescence of Trp-214 in gHSA is 0.72 of that of HSA and the far-UV CD spectrum of gHSA is nearly identical to that of HSA. These results showed that glycation altered the local structure around Trp-214 while not significantly impacting the secondary structure, and this alteration translated into an overall change in the stability of gHSA compared to HSA.     

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 10⁴ M^?1implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.     

Energetics of the interactions of human serum albumin with cationic surfactant

The heat capacity changes for interaction of human serum albumin (HSA) and a cationic surfactant—cetylpyridinium chloride (CPC), were studied at conditions close to physiological (50 mM HEPES or phosphate buffer, pH 7.4 and 160 mM NaCl) carrying out isothermal calorimetric titrations (ITC) at various temperatures (20-40 °C). ITC measurements indicated that the small endothermic changes associated with CPC demicellization were temperature independent at these conditions. Surprisingly, important enthalpy changes associated with binding of CPC to HSA were exothermic and temperature independent at lower concentrations (below 0.022 mM) of CPC and endothermic and temperature dependent at higher concentrations of CPC. The values of heat capacity changes were obtained for each studied concentration of CPC from the plot of enthalpy changes vs temperature. The obtained results demonstrate the temperature independence of heat capacity changes at entire range of studied CPC concentrations. Both enthalpograms and heat capacity curves indicate the two-step mechanism of HSA folding changes due to its interactions with CPC. The first step corresponds to transition from native state to partially unfolded state and the second to unfolding and to the loss of tertiary structure. The analysis of the results indicates that predominant cooperative unfolding occurs at CPC/HSA molar ratio region between 25 and 30. Such information could not be extracted from thermograms and describes the role of heat capacity as a major thermodynamic quantity giving insight on physical, mechanistic and even atomic-level into how HSA may unfold and interact with CPC. The effect of CPC binding on HSA intrinsic fluorescence, UV-Vis and CD spectra were also examined. Hence, the analysis of spectral data confirms the ITC results about the biphasic mechanism of HSA folding changes induced by CPC. The CD measurement also represents the conservation of considerable secondary structure of HSA due to interaction with CPC.     

Deciphering the binding of carbendazim (fungicide) with human serum albumin: A multi-spectroscopic and molecular modelling studies

Carbendazim is a benzimidazole fungicide used to control the fungal invasion. However, its exposure might lead to potential health problems. The present study evaluates the interaction of carbendazim (CAR) with human serum albumin (HSA) which is an important drug carrier protein and plays a very crucial role in the transportation of small molecules. A number of biophysical techniques were employed to investigate the binding of CAR with HSA. The increased UV-absorption of HSA on titrating with CAR suggests the formation of HSA–CAR complex and it could be due to the exposure of aromatic residues. The fluorescence study confirmed that CAR quenches the fluorescence of HSA and showed the static mode of quenching. CAR (50 µM) quenches around 56.14% of the HSA fluorescence. The quenching constant, binding constant, number of binding site and free energy change was calculated by fluorescence quenching experiment. Competitive displacement assay showed Sudlow’s site I as the primary binding site of CAR on HSA. The synchronous fluorescence study revealed the perturbation in the microenvironment around tyrosine and tryptophan residues upon binding of CAR to HSA. The circular dichroism results suggested that the binding of CAR to HSA altered its secondary structure. Molecular docking experiment demonstrated the binding of CAR to Sudlow’s site I of HSA. Docking studies suggested that the hydrogen bonding, van der Waals and pi-alkyl are playing role in the interaction of CAR with HSA. The study confirmed the conformational changes within HSA upon binding of CAR.     

Studies of phenytoin binding to human serum albumin by high-performance affinity chromatography

High-performance affinity chromatography was used to study the binding of phenytoin to an immobilized human serum albumin (HSA) column. This was accomplished through frontal analysis and competitive binding zonal elution experiments, the latter of which used four probe compounds for the major and minor binding sites of HSA injected into the presence of mobile phases containing known concentrations of phenytoin. It was found that phenytoin can interact with HSA at the warfarin-azapropazone, indole-benzodiazepine, tamoxifen, and digitoxin sites of this protein. The association constants for phenytoin at the indole-benzodiazepine and digitoxin sites were determined to be 1.04 (+/-0.05) x 10(4)M(-1) and 6.5 (+/-0.6) x 10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Both allosteric interactions and direct binding for phenytoin appear to take place at the warfarin-azapropazone and tamoxifen sites. This rather complex binding system indicates the importance of identifying the binding regions on HSA for specific drugs as a means for understanding the transport of such substances in blood and in characterizing their potential for drug-drug interactions.     

The binding constant for amyloid Abeta40 peptide interaction with human serum albumin

Human serum albumin (HSA) is the major carrier of Aβ peptides in blood plasma. 1:1 interaction stoichiometries were established in previous indirect antibody-based studies for both Aβ40 and Aβ42, but corresponding binding constants were not provided. In this study we applied direct titrations of HSA with Aβ40 monitored using circular dichroism spectroscopy and obtained a dissociation constant (K_d) of 5 ± 1 μM for a HSA complex with Aβ40. The interaction resulted in an increase of the α-helical contents in the complex, compared to its components, which is quantitatively consistent with the known ability of Aβ40 to adopt a partially α-helical conformation in a hydrophobic environment. The relevance of these findings for the role of HSA in Aβ physiology is discussed.     

Insights into the binding of paclitaxel to human serum albumin: multispectroscopic studies

The interaction of paclitaxel with human serum albumin (HSA) was studied using fluorescence, resonance light scattering, ultraviolet‐visible, circular dichroism and Fourier transform infrared spectroscopy at pH 7.4. Fluorescence data revealed that the fluorescence quenching of HSA by paclitaxel was a static quenching procedure. Time‐resolved fluorescence data also confirmed the quenching mode, which present a constant decay time of about 5 ns. The binding sites were approximately 1 and the binding constant suggested a weak association (324/M at 298 K), which is helpful for the release of the drug to targeted organs. The thermodynamic parameters, ΔG^○, ΔH° and ΔS° were calculated as – 1.06 × 10⁴ J/mol, 361 J/mol per K and 9.7 × 10⁴ J/mol respectively at 298 K, suggesting that binding was spontaneous and was driven mainly by hydrophobic interactions. The binding distance between HSA and paclitaxel was determined to be 2.23 nm based on the Förster theory. Analysis of circular dichroism, ultraviolet‐visible, three‐dimensional fluorescence, Fourier transform infrared and resonance light scattering spectra demonstrated that HSA conformation was slightly altered in the presence of paclitaxel and dimension of the individual HSA molecules were larger after interacting with paclitaxel. These results were confirmed by a molecular docking study. Copyright © 2013 John Wiley & Sons, Ltd.     

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van’t Hoff plots. An enthalpy–entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy–Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.     

New mathematical approach for the evaluation of drug binding to human serum albumin by high-performance liquid affinity chromatography

A novel mathematical approach for investigation of drug–human serum albumin (HSA) interactions by means of high-performance liquid affinity chromatography is developed. The model is based on the assumption that two types of competitive binding sites exist on the HSA molecule. The widely used single-site binding equation is extended and a proper mathematical analysis is proposed allowing the determination of the major parameters characterizing the multisite binding (cobinding) process. The utility of the new approach is proved by competitive studies on HSA binding of two model drugs, diazepam and diclofenac.     

Comparative studies on the interactions of dihydroartemisinin and artemisinin with bovine serum albumin using spectroscopic methods

The interactions of dihydroartemisinin (DHA) and artemisinin (ART) with bovine serum albumin (BSA) have been investigated using fluorescence, UV/vis absorption and Fourier transform infrared (FTIR) spectra under simulated physiological conditions. The binding characteristics of DHA/ART and BSA were determined by fluorescence emission and resonance light scattering (RLS) spectra. The quenching mechanism between BSA and DHA/ART is static. The binding constants and binding sites of DHA/ART–BSA systems were calculated at different temperatures (293, 298, 304 and 310 K). According to Förster non‐radiative energy transfer theory, the binding distance of BSA to DHA/ART was calculated to be 1.54/1.65 nm. The effect of DHA/ART on the secondary structure of BSA was analyzed using UV/vis absorption, FTIR, synchronous fluorescence and 3D fluorescence spectra. In addition, the effects of common ions on the binding constants of BSA–DHA and BSA–ART systems were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Fatty acid binding to serum albumin: Molecular simulation approaches

Background

Binding affinity for human serum albumin (HSA) is one of the most important factors affecting the distribution and free blood concentration of many ligands. The effect of fatty acids (FAs) on HSA-ligand binding has long been studied. Since the elucidation of the 3-dimensional structure of HSA, molecular simulation approaches have been applied to studies of the structure–function relationship of HSA–FA binding.

Scope of review

We review current insights into the effects of FA binding on HSA, focusing on the biophysical insights obtained using molecular simulation approaches such as docking, molecular dynamics (MD), and binding free energy calculations.

Major conclusions

Possible conformational changes on binding of FA molecules to HSA have been observed through MD simulations. High- and low-affinity FA-binding sites on HSA have been identified based on binding free energy calculations. The relationship between the warfarin binding affinity of HSA and FA molecules has been clarified based on the results of simulations of multi-site FA binding that cannot be experimentally observed.

General significance

Molecular simulation approaches have great potentials to provide detailed biophysical insights into HSA as well as the effects of the binding of FAs or other ligands to HSA. This article is part of a Special Issue entitled Serum Albumin.     

New insight into the stereoselective interactions of quinine and quinidine,with bovine serum albumin

Quinine (QN) and quinidine (QD), the chief quinoline alkaloids of various species of cinchona bark, are stereoisomers to each other. In this study, a series of appropriate and efficient methods have been applied to compare the binding modes of QN and QD with bovine serum albumin (BSA). The isothermal titration calorimetry and room temperature phosphorescence results show that both QN and QD can interact with BSA at one binding site to form drug–protein complexes, mainly through enthalpic driving force with the binding affinity order: QN > QD. The fluorescence resonance energy transfer and time‐resolved fluorescence spectroscopy exhibits that QN has a larger energy transfer and more intensified binding capacity for BSA than QD. Data of dynamic light scattering reveal that the aggregate state of BSA is changed during this binding process, and the particle size distribution of QN‐BSA bioconjugate is larger than that of QD. Nuclear magnetic resonance analysis indicates that aromatic protons make more contribution during ligand‐protein complexation than that of aliphatic protons. The circular dichroism spectra exhibit different degrees of changes in BSA secondary structures in the presence of QN and QD, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Interaction of copper (II) complexes by bovine serum albumin: spectroscopic and calorimetric insights

Serum albumins being the most abundant proteins in the blood and cerebrospinal fluid are significant carriers of essential transition metal ions in the human body. Studies of copper (II) complexes have gained attention because of their potential applications in synthetic, biological, and industrial processes. Study of binding interactions of such bioinorganic complexes with serum albumins improves our understanding of biomolecular recognition process essential for rational drug design. In the present investigation, we have applied quantitative approach to explore interactions of novel synthesized copper (II) complexes viz. [Cu(L¹)(L²)ClO₄] (complex I), [Cu(L²)(L³)]ClO₄] (complex II) and [Cu(L⁴)₂(H₂O)₂] (complex III) with bovine serum albumin (BSA) to evaluate their binding characteristics, site and mode of interaction. The fluorescence quenching of BSA initiated by complexation has been observed to be static in nature. The binding interactions are endothermic driven by entropic factors as confirmed by high sensitivity isothermal titration calorimetry. Changes in secondary and tertiary structure of protein have been studied by circular dichroism and significant reduction in α-helical content of BSA was observed upon binding. Site marking experiments with warfarin and ibuprofen indicated that copper complexes bind at site II of the protein.