Conformational Changes Of Bovine Serum Albumin Research Articles

Investigation on the interaction of food colorant Sudan III with bovine serum albumin using spectroscopic and molecular docking methods

Sudan III is a coloring agent used in chemical industries and food additives. This article uses spectroscopic and molecular docking methods to investigate the interaction of Sudan III with bovine serum albumin (BSA) under a physiological condition. Spectroscopic analysis of the emission quenching revealed that the quenching mechanism of BSA by Sudan III was static. The binding sites and constants of Sudan III–BSA complex were observed to be from 0.72 and 6.41 × 102 L·mol−1 to 0.69 and 5.83 × 102 L·mol−1 at 298 and 310 K, respectively. The enthalpy change (ΔH) and entropy change (ΔS) revealed that van der Waals forces and hydrogen bonds stabilized the Sudan III–BSA complex. Energy transfer from tryptophan to Sudan III occurred by a fluorescence resonance energy transfer mechanism, and the r distance (3.32 nm) had been determined. The results of UV–Vis absorption, synchronous, three-dimensional fluorescence, and circular dichroism spectra showed that Sudan III induced conformational changes of BSA. Molecular docking studies revealed that Sudan III situated within subdomain IIA of BSA. A study on the interaction between Sudan III and BSA was of fundamental importance for providing more information about the potential toxicological effect of chemicals at the molecular level.

Interaction of tebuconazole with bovine serum albumin: determination of the binding mechanism and binding site by spectroscopic methods

This study investigates the interaction between tebuconazole and bovine serum albumin (BSA) in a physiological buffer (pH = 7.4) using the fluorescence quenching method to obtain the apparent binding constants (K) and number of binding sites (n) in the interaction between tebuconazole and BSA. The results revealed that tebuconazole can quench the intrinsic fluorescence of BSA through a static quenching procedure. It also shows that the thermodynamic parameters of enthalpy change (ΔH) and entropy change (ΔS) are negative, indicating that the interaction of tebuconazole with BSA is mainly driven by van der Waals forces and hydrogen bonds. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance of r between the donor (BSA) and acceptor (tebuconazole) was calculated to be 0.68 nm based on Forster’s non-radiative energy transfer theory. Analysis of synchronous fluorescence, three-dimensional fluorescence and circular dichroism (CD) spectra demonstrates that tebuconazole can induce conformational changes of BSA.

Investigation of the Interaction of Chrysene and Bovine Serum Albumin by Multispectroscopic Method

The interaction mechanisms of chrysene with bovine serum albumin (BSA) were studied by multispectroscopy and molecular docking method. The quenching mechanism, binding constants, thermodynamic parameters and binding force of chrysene with BSA were investigated. The results indicated that the fluorescence of BSA was quenched by chrysene through a static quenching procedure. The binding constants K b and number of binding sites n were 3.97 × 104 M–1 and 1.0 at 298 K, respectively, which indicated that the binding of chrysene and BSA was moderate and there was one main binding site in BSA for chrysene. The thermodynamic parameters including entropy change (ΔH) and enthalpy change (ΔS) were 40.2 kJ mol–1 and 223.0 J mol–1 K–1, respectively, which revealed that the main binding force of chrysene to BSA was hydrophobic interaction. This result was in accordance with the results from the molecular docking. The binding distance r was 2.60 nm, which suggested that the energy transfer from BSA to chrysene occurred with high possibility. The results of synchronous fluorescence spectra, three-dimensional fluorescence spectra and circular dichroism spectra indicated that the binding of chrysene to BSA induced conformation changes of BSA.

Conformation changes of albumin and lysozyme on electrospun TiO2 nanofibers and its effects on MSC behaviors

Protein adsorption plays a key role in bone repair and regeneration by affecting cell behavior. In this study, TiO2 nanofibers (TiO2 NFs) with different structures, including anatase TiO2 nanofibers (A-NFs), anatase TiO2 nanofibers with beads (B-NFs), anatase-rutile TiO2 nanofibers (AR-NFs) and rutile TiO2 nanofibers (R-NFs), were prepared by electrospinning method. Bovine serum albumin (BSA) and lysozyme (LYZ) were used to explore the adsorption behaviors of TiO2 NFs and then the effects of materials with protein on bone marrow mesenchymal stem cells (MSCs) were studied. Pure titanium metal (PT) was used as control. The results displayed that the adsorption amounts of BSA on samples were B-NFs > AR-NFs > A-NFs ≈ R-NFs > PT, and that for LYZ were B-NFs > AR-NFs > R-NFs > A-NFs > PT. The conformation of proteins changed remarkably when they were adsorbed on meterials. Soaking the TiO2 NFs with and without protein into SBF revealed that the BSA and LYZ on B-NFs, A-NFs and AR-NFs could accelerate the HA deposition on its surface, but it had no promoting effect on HA deposition on B-NFs. MTT and PCR tests showed that the BSA and LYZ adsorbed on materials could promote the proliferation and osteogenic differentiation of MSCs to different degrees due to their different adsorption amount and conformation changes on different TiO2 NFs. The current work demonstrated that the surface properties and crystal structure of TiO2 NFs could influence the adsorption behavior and conformational change of BSA and LYZ, and then further regulate MSCs biological behavior.

Effect of adiphenine hydrochloride on the structure of bovine serum albumin: Spectroscopic and docking study

The interaction of adiphenine hydrochloride (ADP) with bovine serum albumin (BSA) has been investigated by fluorescence, time resolved fluorescence, UV–visible, FTIR and tensiometric technique. Molecular docking has also been done to show the available binding sites on BSA for ADP. Critical micelle concentration (cmc), surface excess (Γmax), the minimum area per molecule (Amin), and molar free energy at the maximum adsorption attained at cmc (Gmin) were evaluated to study the interfacial properties of ADP. Fluorescence spectroscopy was used to observe the effective quenching mechanism between BSA and ADP which was further confirmed by time-resolved fluorescence spectroscopy. Different thermodynamic parameters such as molar enthalpy (ΔH), molar Gibbs free energy (ΔG), and molar entropy contribution (ΔS) were evaluated by using van’t Hoff equation which showed that the binding mechanism was enthalpy driven and the major forces involved between ADP and BSA interaction are van der Waals and hydrogen bonding. UV–vis spectroscopy results showed the complex formation between ADP and BSA. Synchronous fluorescence spectra reveal that there is a slight effect of ADP on the fluorescence intensity of tyrosine but a significant effect on tryptophan residue of BSA. FTIR results suggested the ADP induced conformational changes in BSA. In addition, the molecular docking and competitive binding results showed that the ADP molecule binds with BSA on Sudlow site I (subdomain IIA). The esterase-like enzymatic activity study revealed that the ADP inhibits the enzymatic activity of BSA.

Bovine Serum Albumin Amplified Reactive Oxygen Species Generation from Anthrarufin-Derived Carbon Dot and Concomitant Nanoassembly for Combination Antibiotic–Photodynamic Therapy Application

Amplification of reactive oxygen species (ROS) generation through covalent conjugation of bovine serum albumin (BSA) with newly synthesized, ROS-producing carbon dots (CDs) upon visible light irradiation is reported for the first time. Derivatization of surface carboxyl functional groups of Anthrarufin-derived, green-emitting CD with the amine functionality of BSA ushers distinct changes in the photophysics of CD including an unprecedented ∼50 nm shift in its excitation maxima, decrease in fluorescence lifetime, and concomitant increase in ROS generation. Substantial conformational changes of BSA were witnessed upon conjugation with CD, rendering the BSA-CD conjugate resistant to pepsinolysis. A protease-proof nanoassembly was derived from the BSA-CD conjugate through desolvation that simultaneously hosts a prototype antibiotic and generates ROS with excellent efficiency, making it an attractive platform for antibacterial photodynamic therapy (A-PDT) applications. Systemic annihilation of both Gram-positive and -negative bacteria was achieved with the BSA-CD nanoassembly and envisioned as alternatives to traditional photosensitizers.

BSA binding and antibacterial studies of newly synthesized 5,6-Dihydroimidazo[2,1-b]thiazole-2-carbaldehyde.

A new heterocyclic compound, 5,6-Dihydroimidazo[2,1-b]thiazole-2-carbaldehyde (ITC) was synthesized and its antibacterial activity and also its interaction with bovine serum albumin (BSA) were studied. The structure of the synthesized compound was confirmed by 1H NMR, 13C NMR and IR spectroscopic techniques. The antibacterial activity was carried out by minimum inhibitory concentration (MIC) method. The compound showed a good antibacterial activity. The mechanism of interaction between the BSA and ITC under physiological conditions was investigated by various molecular spectroscopic techniques like, fluorescence, circular dichroism (CD), UV absorption and FT-IR. The interaction between ITC and BSA was followed by studying the quenching of intrinsic fluorescence of BSA upon the addition of ITC at three different temperatures. The binding constant (K), Stern-Volmer quenching constant (Ksv) and number of binding sites were determined. The separation distance between BSA and ITC was evaluated based on the fluorescence resonance energy transfer theory. The conformational changes in BSA upon binding of ITC were also confirmed. The interference of some metal ions on interaction was studies. The displacement studies with site specific markers confirm that the site III was the binding site for ITC on BSA.

The effect of Pseudomonas fluorescens biosurfactant pseudofactin II on the conformational changes of bovine serum albumin: Pharmaceutical and biomedical applications

Herein, we have investigated the binding interaction of bovine serum albumin (BSA) with a biosurfactant pseudofactin II (PF) in physiological buffer medium through the use of surface tension, fluorescence, circular dichroism (CD), isothermal titration calorimetry (ITC) and molecular docking studies. Fluorescence results showed that PF interacts with BSA through a static quenching mechanism. CD results demonstrate a conformational change in BSA upon PF combination. The negative Gibbs free energies obtained through ITC experiment suggest that the binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process, showing a binding energy of −58.6 kcal mol−1. The present study will help understand the binding mechanism of lipopeptide biosurfactant (PF) to proteins as well as the associated stability and conformational changes.

MWCNT interactions with protein: surface-induced changes in protein adsorption and the impact of protein corona on cellular uptake and cytotoxicity.

PurposeProtein adsorption onto nanoparticles in the form of protein corona, affects properties of nanomaterials and their behavior in the biological milieu. This study aims at exploring the effects of multiwalled carbon nanotubes (MWCNTs) surface chemistry on bovine serum albumin (BSA) and immunoglobulin G (IgG), including their adsorption behavior and spatial configurations, as well as the impact on cellular uptake, cytotoxicity, and cellular responses.MethodsThree types of MWCNTs (pristine MWCNTs, MWCNTs-COOH, and MWCNTs-PEG) were synthesized by classical chemical reduction. The size, morphology, hydrodynamic size, and zeta potential were characterized using transmission electron microscopy and dynamic light scattering. MWCNTs were exposed to BSA and IgG solutions, then the amount of MWCNT absorption was performed by bicinchoninic acid assay, and the effects were assessed by utilizing fluorescence spectroscopy, circular dichroism (CD) spectroscopy. Quantitative measurement of MWCNTs uptake with or without protein corona was performed as turbidity method. CCK assay and a microdilution method were performed to evaluate the effects of protein corona on cytotoxicity and pro-inflammatory cytokines release.ResultsThe BSA and IgG adsorption capacities of MWCNTs followed the order pristine MWCNTs>MWCNTs-COOH and MWCNTs-PEG. MWCNT binding can cause fluorescence quenching and conformational changes in BSA and IgG, indicating that both the physicochemical properties of MWCNTs and protein properties play critical roles in determining their adsorption behavior. Further study showed time-dependent increases in MWCNT cellular uptake and internalization. Hydrophobicity is the major factor increasing cellular uptake of pristine MWCNTs, but a protein corona enriched with dysoposnins is the main factor reducing uptake of MWCNT-COOH by RAW264.7 cells. The cytotoxicity and pro-inflammatory response related to physicochemical properties of MWCNTs, and frustrated phagocytosis is a key initiating event in the pro-inflammatory response of MWCNT-exposed macrophages.ConclusionThese findings shed light on how functionalized MWCNTs interact with protein coronas and provide useful insight into the dramatic effect of protein coronas on different functionalized MWCNTs. These events affect cellular uptake and cytotoxicity, which could inform how to enhance MWCNT biocompatibility and develop approaches for managing MWCNT hazards.

Mechanistic Interaction Study of Bromo-Noscapine with Bovine Serum Albumin employing Spectroscopic and Chemoinformatics Approaches

Bromo-Noscapine (BrNs) is a tubulin-binding cytotoxic agent with significant activity against breast and lung cancer. The mechanistic interaction insight into the binding of bovine serum albumin (BSA) with BrNs can provide critical information about the pharmacodynamics and pharmacokinetics properties. Here, various spectroscopic techniques and computational methods were employed to understand the dynamics of BrNs and BSA interaction. The intrinsic fluorescence of BSA was quenched by BrNs through a static quenching procedure. The stoichiometry of BrNs-BSA complex was 1:1 and binding constant of the complex was in the order of 103 M−1 at 298 K. Based on thermodynamic analysis, it was deduced that binding process of the BrNs with BSA was spontaneous and exothermic, and the major forces between BrNs and BSA were van der waals forces and hydrogen bonding. Moreover, results of FT-IR, CD, UV spectra concluded significant conformational change in BSA on binding with BrNs. The in vitro findings were further confirmed by in silico assays. Molecular docking showed strong interactions with score of −8.08 kcal/mol. Molecular dynamics simulation analysis also suggested the stable binding with lower deviation in RMSD and RMSF values through persistent long simulation run. This study suggests optimal efficiency of diffusion of the BrNs into the bloodstream for the treatment of cancer.

Characterization of the binding and conformational changes of bovine serum albumin upon interaction with antihypertensive olmesartan medoxomil

The interaction of bovine serum albumin (BSA) with the olmesartan medoxomil (OLM) was investigated by different spectroscopic and linear sweep voltammetric techniques under experimentally optimised physiological conditions. The alteration of functional properties of BSA when quenched by OLM was illustrated by the continuous decrease of the fluorescence intensity of BSA upon addition of OLM. The quenching constants (Ksv) obtained were 0.71 × 104, 1.3 × 104 and 2.1 × 104 Lmol−1 at 288, 298 and 308 K respectively. The binding mechanism was dynamic type quenching. The value of K is of the order of 104, indicating that a long-lasting interaction exists between OLM and BSA. The positive values of ΔH0 and ΔS0 suggested the hydrophobic interactions play the major roles in the binding reaction. The conformational change of BSA after binding with OLM was demonstrated by the synchronous, FTIR and 3-D fluorescence spectral results. The effects of some common metal ions and site probes on binding of BSA– OLM were also investigated.

Comparative study of the interactions between bisphenol-A and its endocrine disrupting analogues with bovine serum albumin using multi-spectroscopic and molecular docking studies

Interaction studies of bisphenol analogues; biphenol-A (BPA), bisphenol-B (BPB), and bisphenol-F (BPF) with bovine serum albumin (BSA) were performed using multi-spectroscopic and molecular docking studies at the protein level. The mechanism of binding of bisphenols with BSA was dynamic in nature. SDS refolding experiments demonstrated no stabilization of BSA structure denatured by BPB, however, BSA denatured by BPA and BPF was found to get stabilized. Also, CD spectra and molecular docking studies revealed that BPB bound more strongly and induced more conformational changes in BSA in comparison to BPA. Hence, this study throws light on the replacement of BPA by its analogues and whether the replacement is associated with a possible risk, raising a doubt that perhaps BPB is not a good substitute of BPA.

Interactions of pyrene and/or 1-hydroxypyrene with bovine serum albumin based on EEM-PARAFAC combined with molecular docking

The interactions of pyrene (Pyr) and/or 1-hydroxypyrene (1-OHPyr) with bovine serum albumin (BSA) in binary and ternary systems were investigated using the excitation-emission matrix (EEM)-parallel factor analysis (PARAFAC) method combined with fluorescence quenching analysis and the molecular docking method. The results showed that the PARAFAC approach could be used to decompose the EEM spectra of Pyr, 1-OHPyr, and BSA in the binary and ternary systems. The binding constants of Pyr and 1-OHPyr with BSA increased from 1.01 × 106 and 1.62 × 106 L mol−1 to 2.09 × 106 and 1.86 × 107 L mol−1 in the ternary systems compared with the binary systems, respectively. Molecular docking revealed that in both binary and ternary systems, Pyr was bound between II A and III A regions of BSA, whereas 1-OHPyr was located in the I B region. Van der Waals forces dominated the formation of the BSA-Pyr complexes; however, for BSA-1-OHPyr complexes, in addition to Van der Waals forces, hydrogen bonds also played an important role in their binding as a hydrogen bond formed between 1-OHP and the amino residue of BSA. Moreover, the coexistence of Pyr and 1-OHPyr aggravated the conformation changes of BSA and led to a prominent decrease in the hydrophobicity of the micro-environment around tryptophan (TRP) residues. 1-OHPyr has a more severe influence on BSA conformation than Pyr in the ternary systems. This study will help to understand the combined effects of PAHs and their hydroxyl metabolites on proteins at the molecular level.

Size-dependent binding of pristine fullerene (nC60) nanoparticles to bovine/human serum albumin

The size of nC60 nanoparticles may exert profound influence on their binding to serum albumins and their ability to alter protein structure and conformation. To verify this speculation, in this work, five fractions of nC60 dispersions with different particle size distributions were prepared by centrifuge method and characterized using UV–Visible absorption spectroscopy and transmission electronic microscopy. In this work, the binding of five fractions of nC60 nanoparticles with bovine serum albumin (BSA) was demonstrated using fluorescence and synchronous fluorescence spectroscopy. Through the analysis and comparison of binding interactions, it was found that the diameter of nC60 nanoparticles plays a key role in the binding patterns of nC60 nanoparticles to BSA. The smaller nC60 nanoparticles had stronger binding to BSA and exhibited greater influence on the conformation change of BSA. Further study showed that human serum albumin (HSA) has similar binding interaction to nC60 nanoparticles, but the conformation changes of human serum albumin caused by five fractions of nC60 nanoparticles were different from bovine serum albumin. Despite the different particle sizes, the binding sites of nC60 nanoparticles to BSA/HSA were mainly in the vicinity of tryptophan residues. The possible interaction mechanism and the prospects were also discussed.

Intermolecular interaction of fosinopril with bovine serum albumin (BSA): The multi-spectroscopic and computational investigation.

The intermolecular interaction of fosinopril, an angiotensin converting enzyme inhibitor with bovine serum albumin (BSA), has been investigated in physiological buffer (pH7.4) by multi-spectroscopic methods and molecular docking technique. The results obtained from fluorescence and UV absorption spectroscopy revealed that the fluorescence quenching mechanism of BSA induced by fosinopril was mediated by the combined dynamic and static quenching, and the static quenching was dominant in this system. The binding constant, Kb , value was found to lie between 2.69×103 and 9.55×103 M-1 at experimental temperatures (293, 298, 303, and 308K), implying the low or intermediate binding affinity between fosinopril and BSA. Competitive binding experiments with site markers (phenylbutazone and diazepam) suggested that fosinopril preferentially bound to the site I in sub-domain IIA on BSA, as evidenced by molecular docking analysis. The negative sign for enthalpy change (ΔH0 ) and entropy change (ΔS0 ) indicated that van der Waals force and hydrogen bonds played important roles in the fosinopril-BSA interaction, and 8-anilino-1-naphthalenesulfonate binding assay experiments offered evidence of the involvements of hydrophobic interactions. Moreover, spectroscopic results (synchronous fluorescence, 3-dimensional fluorescence, and Fourier transform infrared spectroscopy) indicated a slight conformational change in BSA upon fosinopril interaction.

Molecular interactions of thymol with bovine serum albumin: Spectroscopic and molecular docking studies.

Thymol is the main monoterpene phenol present in the essential oils which is used in the food industry as flavoring and preservative agent. In this study, the interaction of thymol with the concentration range of 1 to 6μM and bovine serum albumin (BSA) at fixed concentration of 1μM was investigated by fluorescence, UV-vis, and molecular docking methods under physiological-like condition. Fluorescence experiments were performed at 5 different temperatures, and the results showed that the fluorescence quenching of BSA by thymol was because of a static quenching mechanism. The obtained binding parameters, K, were in the order of 104 M-1 , and the binding number, n, was approximately equal to unity indicating that there is 1 binding site for thymol on BSA. Calculated thermodynamic parameters for enthalpy (ΔH), entropy (ΔS), and Gibb's free energy (ΔG) showed that the reaction was spontaneous and hydrophobic interactions were the main forces in the binding of thymol to BSA. The results of UV-vis spectroscopy and Arrhenius' theory showed the complex formation in the interaction of thymol and BSA. Negligible conformational changes in BSA by thymol were observed in fluorescence experiments, and the same results were also obtained from UV-vis studies. Results of molecular docking indicated that the subdomain IA of BSA was the binding site for thymol.

Elucidating the interaction of ticlopidine with serum albumin and its role in bilirubin displacement in vitro

Ticlopidine is an anti-platelet drug that functions as a P2Y12 receptor antagonist. The present study provides a detailed characterization of interaction of ticlopidine with a model transport protein, bovine serum albumin (BSA) as well as an assessment of its bilirubin displacing ability using a multi-spectroscopic approach in combination with isothermal titration calorimetry. The value of binding constant determined using ITC studies was found to be 3.03 × 103 M−1 with a binding stoichiometry of approximately 1:1. Competitive site marker experiments indicate that ticlopidine binds to Sudlow site I, located in subdomain IIA of BSA. In addition, Circular dichroism and 3D fluorescence spectroscopy indicated structural and conformational changes in BSA on interaction with ticlopidine. Thermodynamic parameters suggested that the reaction was spontaneous, exothermic, entropically driven, and involved hydrophobic interactions. These results were well supported by those obtained through molecular docking studies. Additionally, the effect of ticlopidine on bilirubin and albumin interaction was evaluated using the peroxidase method as well as through fluorescence spectroscopy. Ticlopidine was found to displace bilirubin from serum albumin. Moreover, the binding constant of bilirubin–serum albumin interaction also decreased in presence of ticlopidine. The results indicated that ticlopidine is a competitive displacer of bilirubin in vitro and may contribute to the incidences hyperbilirubinemia associated with the usage of this drug.

Template free solvothermal synthesis of single crystal magnetic Fe3O4 hollow spheres, their interaction with bovine serum albumin and antibacterial activities

Uniform sized single crystal magnetic Fe3O4 hollow spheres (MFHS) have been synthesized through simple solvothermal method using ferric chloride hexahydrate and 1,3-propanediamine. The reaction time and the amount of 1,3-propanediamine play major roles in the formation of magnetic Fe3O4 hollow spheres. The synthesized products are characterized using X-ray diffraction, scanning electron microscopy, transmission electron microscopy and vibrating sample magnetometry techniques. The crystalline Fe3O4 materials are composed of well-aligned hollow sphere magnetite nanoparticles and exhibit high saturation magnetization of 57.9 emu/g and a remnant magnetization of 17.6 emu/g at room temperature. MFHS interact strongly with bovine serum albumin (BSA) and brings out considerable conformational changes in BSA as evident from the UV–vis absorption, fluorescence and circular dichroism (CD) spectroscopic studies pertaining to the interaction of synthesized MFHS and BSA. The prepared MFHS effectively inhibit the growth of Pseudomonas aeruginosa and Staphylococcus aureus.

Physicochemical study of biomolecular interactions between lysosomotropic surfactants and bovine serum albumin

The interactions between two cationic lysosomotropic surfactants (2-dodecanoyloxyethyl)trimethylammonium bromide (DMM-11) and (2-dodecanoyloxypropyl)trimethylammonium bromide (DMPM-11) with bovine serum albumin (BSA) in Hepes buffer (pH=7.4) were systematically studied by surface tension, fluorescence and circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). Furthermore, the size of the micellar aggregates and the polydispersity indexes of both cationic surfactants were studied by dynamic light scattering technique (DLS). The hydrodynamic radii, micellar volumes and aggregation numbers were calculated using a method based on density functional theory (DFT). The results showed that, in both cases, the surface tension was modified upon addition of BSA, and the critical micelle concentration (CMC) values of DMM-11 and DMPM-11 were higher in the presence of BSA. The fluorescence intensity of BSA decreased significantly as the concentration of both cationic surfactants increased and this effect was attributed to the formation of surfactant-BSA complexes. Synchronous fluorescence spectrometry showed the binding-induced conformational changes in BSA. Finally, CD and DLS results revealed the occurrence of changes in the secondary structure of the protein in the presence of both surfactants. In conclusion, understanding the interactions between lysosomotropic surfactants and BSA is required to explore their potential applications in medicine.

Multi-spectroscopic and molecular modeling approaches to elucidate the binding interaction between bovine serum albumin and darunavir, a HIV protease inhibitor

Darunavir (DRV), a second-generation HIV protease inhibitor, is widely used across the world as an important component of HIV therapy. The interaction of DRV with bovine serum albumin (BSA), a major carrier protein, has been studied under simulated physiological conditions (pH7.4) by multi-spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the intrinsic fluorescence of BSA was quenched by DRV in terms of a static quenching procedure due to the formation of the DRV-BSA complex. The results indicated the presence of single weak affinity binding site (~103M−1, 310K) on protein. The thermodynamic parameters, namely enthalpy change (ΔH0), entropy change (ΔS0) and Gibbs free energy change (ΔG0) were calculated, which signified that the binding reaction was spontaneous, the main binding forces were hydrogen bonding and van der Waals forces. Importantly, competitive binding experiments with three site probes, phenylbutazone (in sub-domain IIA, site I), ibuprofen (in sub-domain IIIA, site II) and artemether (in the interface between sub-domain IIA and IIB, site II’), suggested that DRV was preferentially bound to the hydrophobic cavity in site II’ of BSA, and this finding was validated by the docking results. Additionally, synchronous fluorescence, three-dimensional fluorescence and Resonance Rayleigh Scattering (RRS) spectroscopy gave qualitative information on the conformational changes of BSA upon adding DRV, while quantitative data were obtained with Fourier transform infrared spectroscopy (FT-IR).