Direct interactions in the recognition between the environmental estrogen bisphenol AF and human serum albumin

Bisphenol AF (BPAF) was used as a model compound to investigate the binding mechanism between the endocrine disrupting compound and human serum albumin (HSA) using multispectroscopic techniques and molecular modeling method at the protein level. The results indicated that BPAF was indeed bound to HSA and located in the hydrophobic pocket of HSA on subdomain IIA through hydrogen bond and van der Waals interactions. The fluorescence quenching data showed that the binding of BPAF and HSA quenched the intrinsic fluorescence of HSA, and the static quenching constants were acquired. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

The nature of the hydrophobic binding of small peptides at the bilayer interface: implications for the insertion of transbilayer helices

One method of obtaining useful information about the physical chemistry of peptide/bilayer interactions is to relate thermodynamic parameters of the interactions to structural parameters obtained by diffraction methods. We report here the results of the application of this approach to interactions of hydrophobic tripeptides of the form Ala-X-Ala-O-tert-butyl with lipid bilayers. The thermodynamic constants (delta Gt, delta Ht, and delta St) for the transfer of the tripeptides from water into DMPC vesicles were determined for X = Leu, Phe, and Trp and found to be consistent with those expected for hydrophobic interactions above the phase transition of DMPC. Combining these results with the earlier ones of Jacobs and White [(1986) Biochemistry 25, 2605-2612], the favorable free energies of transfer with different amino acids in the -X- position increase in the order Gly less than Ala less than Leu less than Phe less than Trp in agreement with the Nozaki and Tanford [(1971) J. Biol. Chem. 246, 2211-2217] hydrophobicity scale. Determination of the location of Ala-[2H5]Trp-Ala-O-tert-butyl in oriented DOPC bilayers by neutron diffraction shows that the most hydrophobic peptide of the series is confined to the bilayer headgroup/water region. Refinement of the diffraction measurements shows that only 13% of the tryptophan is associated with the hydrocarbon core. The distribution of the water tends to mirror that of the peptide. Unlike peptide-free bilayers, 5% of the water penetrates the hydrocarbon, which is about 100-fold greater than expected. A quantitative thermodynamic analysis of the interfacial binding of the peptides suggests that (1) the hydrophobic interactions are 60-70% complete upon binding at the bilayer interface, (2) the interface is likely to play an important role in helix formation and insertion, (3) the hydrogen bond status of amino acid side chains is crucial to insertion, and (4) an a priori lack of knowledge of the status of such bonds could limit the precision of hydrophobicity plots. We introduce an interfacial hydrophobicity scale, IFH(h), with a variable hydrogen bond parameter (h) that permits one to consider explicitly hydrogen bonding in transbilayer helix searches.     

Molecular docking,a tool to determine interaction of CuO and TiO2 nanoparticles with human serum albumin

BackgroundWe study the human serum albumin (HSA) protein-CuO nanoparticle interaction to identify the specific binding site of protein with CuO nanoparticles by molecular docking and compared it with HSA-TiO₂ nanoparticle interaction.MethodsThe protein structural data that was obtained using Autodock 4.2.ResultsIn case of CuO np-HSA interaction, the distances from the centre of Subdomain IIIA to Arg-472 is 2.113 Å and Lys 475, Glu 492, Ala 490, Cys 487, Ala 490 are the bound neighbouring residues with Lys 475, Glu 492 at aliphatic region. The binding energy generated was ?1.64 kcal mol^?1. However, for TiO₂ nanoparticle, the binding region is surrounded by Arg 257, Ala 258, Ser 287, His 288, Leu 283, Ala 254, Tyr 150 (subdomain II A) as neighbouring residue. Moreover, Glu 285, Lys 286 forms aliphatic grove for TiO₂-HSA, Ser-287 at the centre region form hydrogen bond with nanoparticle and Leu 283, Leu 284 forming hydrophopobic grove for TiO₂ nanoparticle-HSA interaction. The binding energy generated was ?2.47 kcal mol^?1.ConclusionsAnalysis suggests that CuO bind to suldow site II i.e subdomain III A of HSA protein where as TiO₂ nanoparticle bind to suldow site I i.e subdomain IIA of HSA protein.General significanceThe structural information that derives from this study for CuO and TiO₂ nanoparticles may be useful in terms of both high and low-affinity binding sites when designing these nanoparticles based drugs delivery system.     

Contribution of Gln9 and Phe80 to substrate binding in ribonuclease MC1 from bitter gourd seeds.

Ribonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al. (2000) Biochem. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3',5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased K(m) and 27.6-fold decreased k(cat), while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the k(cat) value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (k(cat)/K(m)) with a minimum K(m) value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2',3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner.     

Saffron carotenoids (crocin and crocetin) binding to human serum albumin as investigated by different spectroscopic methods and molecular docking

Therapeutic effects of saffron ingredients were studied in some diseases. The pharmacokinetics and pharmacodynamics of these ingredients were also studied, but their transport mechanism is not clearly known. Serum albumin has been known as the most important transporter of many drugs in the body that affects their disposition, transportation, and bioavailability. Here, we investigated the interaction of crocin (Cro) with HSA, for the first time, and compared with the crocetin (Crt)–HSA interaction. UV and fluorescence spectroscopy, circular dichroism (CD), and molecular docking was applied to investigate the possibility and mechanism of binding of HSA with these natural carotenoids. The gradually addition of Cro increased HSA absorbency at 278 nm, while Crt decreased it. Both of these changes induced HSA unfolding that was confirmed by the decreased α-helix content, as determined by the CD. Both carotenoids quenched HSA fluorescence emission, but with different mechanisms. The Stern–Volmer plots indicated a dynamic quenching of intrinsic emission of HSA due to Cro addition, while Crt quenching followed both static and dynamic quenching mechanisms. Docking results indicated binding of Cro/Crt in sub-domain IIA, Sudlow site I of HSA, which accompanied with the hydrogen bonding of Cro/Crt with Tyr138. The interaction of these ligands (Cro/Crt) caused HSA unfolding and affects the hydrophobic environment of Trp241, which result in the quenching of Trp fluorescence. The UV spectroscopy and fluorescence quenching data indicated the differences in the mechanisms of interaction of Cro/Crt with HSA, which is due to the differences in the structure and hydrophobicity of these ligands.     

Interaction studies of aristolochic acid I with human serum albumin and the binding site of aristolochic acid I in subdomain IIA

Optical spectroscopy and molecular docking methods were used to examine the binding of aristolochic acid I (AAI) to human serum albumin (HSA) in this paper. By monitoring the intrinsic fluorescence of single Trp214 residue and performing displacement measurements, the specific binding of AAI in the vicinity of Sudlow's Site I of HSA has been clarified. An apparent distance of 2.53 nm between the Trp214 and AAI was obtained via fluorescence resonance energy transfer (FRET) method. In addition, the changes in the secondary structure of HSA after its complexation with the ligand were studied with circular dichroism (CD) spectroscopy, which indicated that AAI does not has remarkable effect on the structure of the protein. Moreover, thermal denaturation experiments clearly indicated that the HSA−AAI complexes are conformationally more stable. Finally, the binding details between AAI and HSA were further confirmed by molecular docking studies, which revealed that AAI was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, van der Waals forces and hydrogen bonding.     

Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme C.

Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 --> Ala, His42 --> Glu variants and the double mutant His42 --> Glu:Arg38 --> Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state.     

Probing the binding mechanism of capecitabine to human serum albumin using spectrometric methods,molecular modeling,and chemometrics approach

Capecitabine as a prodrug of 5-Fluorouracil plays an important role in the treatment of breast and gastrointestinal cancers. Herein, in view of the importance of this drug in chemotherapy, interaction mechanism between Capecitabine (CAP) and human serum albumin (HSA) as a major transport protein in the blood circulatory system has been investigated by using a combination of spectroscopic and molecular modeling methods. The fluorescence spectroscopic results revealed that capecitabine could effectively quench the intrinsic fluorescence of HSA through a static quenching mechanism. Evaluation of the thermodynamic parameters suggested that the binding process was spontaneous while hydrogen bonds and van der Waals forces played a major role in this interaction. The value of the binding constant (K_b = 1.820 × 10⁴) suggested a moderate binding affinity between CAP and HSA which implies its easy diffusion from the circulatory system to the target tissue. The efficiency of energy transfer and the binding distance between the donor (HSA) and acceptor (CAP) were determined according to forster theory of nonradiation energy transfer as 0.410 and 4.135 nm, respectively. Furthermore, UV–Vis spectroscopic results confirmed that the interaction was occurred between HSA and CAP and caused conformational and micro-environmental changes of HSA during the interaction. Multivariate curve resolution-alternating least square (MCR-ALS) methodology as an efficient chemometric tool was used to separate the overlapped spectra of the species. The MCR-ALS result was exploited to estimate the stoichiometry of interaction and to provide concentration and structural information about HSA-CAP interactions. Molecular docking studies suggested that CAP binds mainly to the subdomain IIA of HSA, which were compatible with those obtained by experimental data. Finally, molecular dynamics simulation (MD) was performed on the best docked complex by considering the permanence and flexibility of HSA-CAP complex in the binding site. MD result showed that CAP could steadily bind to HSA in the site I based on the formation of hydrogen bond and π-π stacking interaction in addition to hydrophobic force.     

Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV–vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV–vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.     

Probing the “fingers” domain binding pocket of Hepatitis C virus NS5B RdRp and D559G resistance mutation via molecular docking,molecular dynamics simulation and binding free energy calculations

The NS5B RdRp polymerase is a prominent enzyme for the replication of Hepatitis C virus (HCV). During the HCV replication, the template RNA binding takes place in the “fingers” sub-domain of NS5B. The “fingers” domain is a new emerging allosteric site for the HCV drug development. The inhibitors of the “fingers” sub-domain adopt a new antiviral mechanism called RNA intervention. The details of essential amino acid residues, binding mode of the ligand, and the active site intermolecular interactions of RNA intervention reflect that this mechanism is ambiguous in the experimental study. To elucidate these details, we performed molecular docking analysis of the fingers domain inhibitor quercetagetin (QGN) with NS5B polymerase. The detailed analysis of QGN-NS5B intermolecular interactions was carried out and found that QGN interacts with the binding pocket amino acid residues Ala97, Ala140, Ile160, Phe162, Gly283, Gly557, and Asp559; and also forms π?π stacking interaction with Phe162 and hydrogen bonding interaction with Gly283. These are found to be the essential interactions for the RNA intervention mechanism. Among the strong hydrogen bonding interactions, the QGN?Ala140 is a newly identified important hydrogen bonding interaction by the present work and this interaction was not resolved by the previously reported crystal structure. Since D559G mutation at the fingers domain was reported for reducing the inhibition percentage of QGN to sevenfold, we carried out molecular dynamics (MD) simulation for wild and D559G mutated complexes to study the stability of protein conformation and intermolecular interactions. At the end of 50?ns MD simulation, the π?π stacking interaction of Phe162 with QGN found in the wild-type complex is altered into T-shaped π stacking interaction, which reduces the inhibition strength. The origin of the D559G resistance mutation was studied using combined MD simulation, binding free energy calculations and principal component analysis. The results were compared with the wild-type complex. The mutation D559G reduces the binding affinity of the QGN molecule to the fingers domain. The free energy decomposition analysis of each residue of wild-type and mutated complexes revealed that the loss of non-polar energy contribution is the origin of the resistance.

Communicated by Ramaswamy H. Sarma     

Synthesis and study on the binding of thiazol‐2(3H)‐ylidine derivative with human serum albumin using spectroscopic and molecular docking methods

In this article, a facile and convenient synthesis of thiazol‐2(3H)‐ylidine derivatives of fatty acid ( 3a – c ) is described. The binding of N′‐(4,5‐dimethyl‐3‐penylthiazol‐2(3H)‐ylidine)octadec‐9‐enehydrazide ( 3a ) with human serum albumin (HSA) is explored using various spectral methods and molecular docking. Fluorescence quenching results show that 3a induces conformational changes in HSA and the polarity around the tryptophan residues is increased. Stern–Volmer quenching plots at different temperatures (298, 305 and 312 K) show that the fluorescence quenching mechanism is static quenching. Synchronous fluorescence, 3D fluorescence spectra, circular dichroism and Fourier transform infrared spectroscopy are used to determine the structural change in HSA on interaction with 3a . Förster resonance energy transfer analysis shows that the binding distance (r₀ = 2.78 nm) between HSA (Trp214) and 3a is within the of range 2–8 nm for quenching to occur. The molecular docking study also confirms that 3a is located in subdomain IIA (site I) of HSA and is stabilized by hydrogen bonding and hydrophobic forces.     

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor.     

Probing the interaction of thionine with human serum albumin by multispectroscopic studies and its in vitro cytotoxic activity toward MCF-7 breast cancer cells

The studies on protein–dye interactions are important in biological process and it is regarded as vital step in rational drug design. The interaction of thionine (TH) with human serum albumin (HSA) was analyzed using isothermal titration calorimetry (ITC), spectroscopic, and molecular docking technique. The emission spectral titration of HSA with TH revealed the formation of HSA–TH complex via static quenching process. The results obtained from absorption, synchronous emission, circular dichroism, and three-dimensional (3D) emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure of HSA. Results from ITC experiments suggested that the binding of TH dye was favored by negative enthalpy and a favorable entropy contribution. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of HSA. Molecular docking study further substantiates that TH binds to the hydrophobic cavity of subdomain IIA (Sudlow site I) of HSA. Further, we have studied the cytotoxic activity of TH and TH–HSA complex on breast cancer cell lines (MCF-7) by MTT assay and LDH assay. These studies revealed that TH–HSA complex showed the higher level of cytotoxicity in cancer cells than TH dye-treated MCF-7 cells and the significant adverse effect did not found in the normal HBL-100 cells. Fluorescence microscopy analyses of nuclear fragmentation studies validate the significant reduction of viability of TH–HSA-treated human MCF-7 breast cancer cells through activation of apoptotic-mediated pathways.     

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.     

Probing the interaction of human serum albumin with bilirubin in the presence of aspirin by multi-spectroscopic, molecular modeling and zeta potential techniques: insight on binary and ternary systems

Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (K(a)) and number of binding site (n) obtained were 1.6 × 10(6)M(-1) and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 10(6)M(-1) and 0.84, respectively, in the presence of ASA (ternary complex) at λ(ex)= 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (C(CIAC)) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.     

Binding mechanism of trans-N-caffeoyltyramine and human serum albumin: Investigation by multi-spectroscopy and docking simulation

trans-N-Caffeoyltyramine (TNC), which was isolated from the Cortex Lycii in our laboratory, is a phenolic amide compound with multiple pharmacological activities. The interaction between TNC and human serum albumin (HSA) was studied by Nuclear magnetic resonance (NMR) relaxation experiment, fluorescence spectroscopy, and docking simulation. NMR methodology is based on the analysis of selective and non-selective spin-lattice relaxation rate enhancements of TNC protons in the presence of the HSA. Result indicated that the interaction occurred between HSA and TNC, and changed the proton magnetic environment of TNC. Fluorescence spectroscopy confirmed that TNC displayed a strong capability to quench the fluorescence of HSA, and the acting forces for binding were hydrogen bonds and van der Waals forces. Furthermore, the circular dichroism, synchronous, and three-dimensional fluorescence spectra, which were employed to determine the conformation of protein, revealed that binding of TNC with HSA could induce conformational changes in HSA. In addition, the molecular modeling results exhibited that TNC mainly bonded to site I in sub-domain IIA of HSA.     

Characterization of human serum albumin's interactions with safranal and crocin using multi-spectroscopic and molecular docking techniques

Interaction mechanisms of human serum albumin (HSA) with safranal and crocin were studied using UV–Vis absorption, fluorescence quenching and circular dichroism (CD) spectroscopies as well as molecular docking techniques. Changes in absorbance and fluorescence of HSA upon interactions with both compounds were attributed to their binding to amino acid chromophores located in subdomains IIA and IIIA. Fluorescence secondary inner filter effect was excluded using 278 nm and 340 nm as the wavelengths of HSA's excitation and fluorescence while safranal and crocin absorbed at 320 nm and 445 nm, respectively. Stern-Volmer model revealed a static quenching mechanism involve the formation of non-fluorescent ground state complexes. Stern-Volmer, Hill, Benesi-Hilbrand and Scatchard models gave apparent binding constants ranged in 4.25 × 10³ - 2.15 × 10⁵ for safranal and 7.67 × 10³ - 4.23 × 10⁵ L mol^?1 for crocin. CD measurements indicated that 13 folds of safranal and crocin unfolded the α-helix structure of HSA by 7.47–21.20%. In-silico molecular docking revealed selective exothermic binding of safranal on eight binding sites with binding energies ranged in ?3.969 to ?6.6.913 kcal/mol. Crocin exothermally bound to a new large pocket located on subdomain IIA (sudlow 1) with binding energy of ?12.922 kcal/mol.These results confirmed the formation of HSA stable complexes with safranal and crocin and contributed to our understanding for their binding characteristics (affinities, sites, modes, forces … etc.) and structural changes upon interactions. They also proved that HSA can solubilize and transport both compounds in blood to target tissues. The results are of high importance in determining the pharmacological properties of the two phytochemical compounds and for their future developments as anticancer, antispasmodic, antidepressant or aphrodisiac therapeutic agents.     

Binding of fluphenazine with human serum albumin in the presence of rutin and quercetin: An evaluation of food‐drug interaction by spectroscopic techniques

The interactions between human serum albumin (HSA) and fluphenazine (FPZ) in the presence or absence of rutin or quercetin were studied by fluorescence, absorption and circular dichroism (CD) spectroscopy and molecular modeling. The results showed that the fluorescence quenching mechanism was static quenching by the formation of an HSA–FPZ complex. Entropy change (ΔS ⁰) and enthalpy change (ΔH ⁰) values were 68.42 J/(mol? K) and ?4.637 kJ/mol, respectively, which indicated that hydrophobic interactions and hydrogen bonds played major roles in the acting forces. The interaction process was spontaneous because the Gibbs free energy change (ΔG ⁰) values were negative. The results of competitive experiments demonstrated that FPZ was mainly located within HSA site I (sub‐domain IIA). Molecular docking results were in agreement with the experimental conclusions of the thermodynamic parameters and competition experiments. Competitive binding to HSA between flavonoids and FPZ decreased the association constants and increased the binding distances of FPZ binding to HSA. The results of absorption, synchronous fluorescence, three‐dimensional fluorescence, and CD spectra showed that the binding of FPZ to HSA caused conformational changes in HSA and simultaneous effects of FPZ and flavonoids induced further HSA conformational changes.     

Phenol red interacts with the protofibril-like oligomers of an amyloidogenic hexapeptide NFGAIL through both hydrophobic and aromatic contacts

Amyloid-associated diseases affect millions of people worldwide. Phenol red exhibits modest inhibition toward fibril formation of human Islet amyloid polypeptide (hIAPP) and its toxicity, which is associated with type II diabetes mellitus. However, the molecular level mechanisms of interactions remain elusive. The binding of phenol red molecules to the protofibrils of an amyloidogenic fragment (NFGAIL) of hIAPP has been investigated by molecular dynamics simulations with explicit solvent. The phenol red molecules were observed to bind primarily along either beta-sheet stacking or beta-strand directions. Through its three aromatic rings, the phenol red molecule preferentially interacted with the hydrophobic side chains of Phe, Leu, and Ile; and the polar sulfone and hydroxyl groups were mainly exposed in solvent. Thus, phenol red improves the solubility of the early protofibrils and represses further growth. Interestingly, there was no obvious preference toward the aromatic Phe residue in comparison to the hydrophobic Leu or Ile residues. The lack of binding along the hydrogen bond direction indicates that phenol red does not directly block the beta-sheet extension. Further free energy analysis suggested that a phenol red analog may potentially improve the binding affinity.