Site Marker Competitive Experiments Research Articles

Investigation of the interaction between S-isoalkyl derivatives of the thiosalicylic acid and human serum albumin

S-isoalkyl derivatives of thiosalicylic acid (isopropyl-(L1), isobutyl-(L2) and isoamyl-(L3)) were selected in order to investigate the binding interaction with the human serum albumin (HSA) using different spectroscopic methods and molecular docking simulation. Association constants and number of binding sites were used to analyze the quenching mechanism. The experimental results showed that the fluorescence quenching of HSA by L1, L2 and L3 occurs because of static quenching and that binding processes were spontaneous, with the leading forces in bonding by hydrogen bonding, hydrophobic interactions, and electrostatic interactions. Fluorescence spectroscopy, UV-Vis spectroscopy and synchronous fluorescence spectroscopy showed that ligands (L1, L2 and L3) can bind to HSA and that the binding of ligands induced some microenvironmental and conformational changes in HSA. The calculated distance between the donor and the acceptor according to fiFörster’s theory confirms the energy transfer efficiency between the acceptor and HSA. Results of site marker competitive experiments showed that the tested compounds bind to HSA in domain IIA (Site I). Molecular dynamics and docking calculations demonstrated that L3 binds to the Sudlow site I of HSA with lower values of binding energies compared to L1 and L2, indicating the formation of the most stable ligand-HSA complex. Understanding the binding mechanisms of S-isoalkyl derivatives of the thiosalicylic acid to HSA may provide valuable data for the future studies of their biological activity and application as potential antitumor drugs. Communicated by Ramaswamy H. Sarma

Mode of binding, kinetic and thermodynamic properties of a lipid-like drug (Fingolimod) interacting with Human Serum Albumin.

Introduction: Fingolimod is a drug that is used to treat multiple sclerosis (MS). It has pH-dependent solubility and low solubility when buffering agents are present. Multi-spectroscopic and molecular modeling methods were used to investigate the molecular mechanism of Fingolimod interaction with human serum albumin (HSA), and the resulting data were fitted to the appropriate models to investigate the molecular mechanism of interaction, binding constant, and thermodynamic properties. Methods: The interaction of Fingolimod with HSA was investigated in a NaCl aqueous solution (0.1 mM). The working solutions had a pH of 6.5. Data was collected using UV-vis, fluorescence quenching titrations, FTIR, and molecular modeling methods. Results: According to the results of the fluorescence quenching titrations, the quenching mechanism is static. The apparent binding constant value (KA = 4.26×103) showed that Fingolimod is a moderate HSA binder. The reduction of the KA at higher temperatures could be a result of protein unfolding. Hydrogen bonding and van der Waals interactions are the main contributors to Fingolimod-HSA complex formation. FTIR and CD characterizations suggested a slight decrease in the α-helix and β-sheets of the secondary structure of HSA due to Fingolimod binding. Fingolimod binds to the binding site II, while a smaller tendency to the binding site I was observed as well. The results of the site marker competitive experiment and the thermodynamic studies agreed with the results of the molecular docking. Conclusion: The pharmacokinetic properties of fingolimod can be influenced by its HSA binding. In addition, considering its mild interaction, site II binding drugs are likely to compete. The methodology described here may be used to investigate the molecular mechanism of HSA interaction with lipid-like drugs with low aqueous solubility or pH-dependent solubility.

Elucidation of the binding mechanism of astragaloside IV derivative with human serum albumin and its cardiotoxicity in zebrafish embryos.

LS-102 is a new derivative of astragaloside IV (AGS IV) that has been shown to possess potentially significant cardioprotective effects. However, there are no reports concerning its interaction with human serum albumin (HSA) and toxicology in vertebrates. The present investigation was undertaken to characterize the interaction of AGS IV and LS-102 with HSA using equilibrium dialysis and UHPLC-MS/MS methods, along with computational methods. Notably, the effects of AGS IV and LS-102 were studied in vivo using the zebrafish embryo model. Markers related to embryonic cardiotoxicity and thrombosis were evaluated. We showed that the plasma protein binding rate of AGS IV (94.04%–97.42%) was significantly higher than that of LS-102 (66.90%–69.35%). Through site marker competitive experiments and molecular docking, we found that AGS IV and LS-102 were located at the interface of subdomains IIA and IIIA, but the site I might be the primary binding site. Molecular dynamics revealed that AGS IV showed a higher binding free energy mainly due to the stronger hydrophobic and hydrogen bonding interactions. Moreover, the secondary structure implied no obvious effect on the protein structure and conformation during the binding of LS-102. LS-102 significantly ameliorated the astramizole-induced heart rate slowing, increased SV-BA spacing, and prevented arachidonic acid-induced thrombosis in zebrafish. To our knowledge, we are the first to reveal that LS-102 binds to HSA with reversible and moderate affinity, indicating its easy diffusion from the circulatory system to the target tissue, thereby providing significant insights into its pharmacokinetic and pharmacodynamic properties when spread in the human body. Our results also provide a reference for the rational clinical application of LS-102 in the cardiovascular field.

A binuclear Cu(I)-phosphine complex as a specific HSA site I binder: synthesis, X-ray structure determination, and a comprehensive HSA interaction analysis

In this research, we present a method for synthesis and a detailed description of geometry characterization of a novel binuclear Cu(I) phosphine complex, along with analysis of its interaction with HSA using spectroscopic and simulation methods. The Cu atoms are coordinated in a tetrahedral geometry, which results in coordination by two nitrogen atoms from the N,N'-(ethane-1,2-diyl)bis(1-(pyridin-2-yl)methanimine ligand (L), a chloride, and a PPh3. The complex binding constant to HSA in a biochemical environment was determined to be ∼106, which is indicative of a strong interaction. The fluorescence of HSA is significantly quenched by binding to the complex via a static mechanism, whereas the microenvironment of the tryptophan residue remains unchanged. A spontaneous binding process was indicated by a negative value for ΔG. Thermodynamic signatures reflect the dominance of hydrophobic forces during the interaction. The site marker competitive experiment combined with docking simulation analysis revealed the closeness position of the complex binding site to warfarin location in specific ligand site I of HSA. The information generated in the present study would be valuable to understand the interaction mechanistic and pharmacological behavior of Cu(I) complexes. Communicated by Ramaswamy H. Sarma

Bovine Serum Albumin as a Potential Carrier for the Protection of Bioactive Quercetin and Inhibition of Cu(II) Toxicity.

Considering the protective ability of proteins and the potential toxicity of free Cu(II), it was proposed herein that the co-presence of protein could play an important role in suppressing the toxicity of free Cu(II) to the stability of bioactive quercetin if a flavonoid-protein-Cu(II) complex could be formed. In this study, the interaction between quercetin (a major flavonoid in the human diet) and bovine serum albumin (BSA) was investigated in the absence and presence of free Cu(II). The results demonstrated that both quercetin and free Cu(II) had a strong ability to quench the intrinsic fluorescence of BSA through a static procedure (i.e., formation of a BSA-monoligand complex). Site marker competitive experiments illustrated that the binding of both quercetin and Cu(II) to BSA mainly took place in subdomain IIA. The quenching process of free Cu(II) with BSA was easily affected by quercetin, and the increased binding capacity possibly resulted from the generation of a ternary quercetin-BSA-Cu(II) complex. The stability and free radical scavenging activity of bioactive quercetin during incubation was promoted in the BSA-diligand complex relative to a quercetin-Cu(II) complex. A quercetin-Cu(II) system could generate reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), which were significantly inhibited upon BSA binding. Consistently, the cytotoxicity of the quercetin-Cu(II) system to endothelial cells was decreased in the BSA-diligand complex, where the co-presence of BSA played an important role. These results suggest the possibility and advantage of developing albumin-based carriers for the protection of bioactive components and suppression of Cu(II) toxicity in their biomedical and nutritional applications.

Synthesis of a IAP antagonist analogue and its binding investigation with BSA/HSA

The development of new drugs especially novel anti-cancer drugs has become a hot-spot issue that catches scholars’ attention in every field. Here a IAP antagonist analogue (IAPA) was synthesized and characterized by NMR and HRMS spectra. Its interaction with proteins, including bovine serum albumin (BSA) and human serum albumin (HSA), were investigated by spectral methods and molecular simulation. Fluorescence quenching, together with UV absorption spectrum and time-resolved fluorescence spectrums indicated a static process in BSA/HSA-IAPA system. Thermodynamic parameters including ΔH and ΔS calculated by Vant't Hoff equation were all positive, showing that hydrophobic force was the major force in the interaction of compound IAPA and BSA/HSA. Site marker competitive displacement experiments proved that IAPA bound to site I at BSA/HSA. Synchronous fluorescence spectrum and three-dimensional fluorescence spectrum were also used to investigate the conformational changes of BSA/HSA after binding with compound IAPA. The above results were further verified by molecular docking.

Study on the cytotoxic, antimetastatic and albumin binding properties of the oxidovanadium(IV) chrysin complex. Structural elucidation by computational methodologies

We have previously synthesized and characterized the chrysin coordination complex with the oxidovanadium(IV) cation (VIVO(chrys)2) and characterized in ethanolic solution and in solid state. Because suitable single crystals for X-ray diffraction determinations could not be obtained, in the present work, we elucidate the geometrical parameters of this complex by computational methodologies. The optimization and vibrational investigation were carried out both in ethanolic solution and in gas phase. The computational results support the experimentally proposed geometries of the VIVO(chrys)2 complex, thus leading to the conclusion that the complex exists as conformers with trans-octahedral geometry in ethanolic solution and as conformers with cis-octahedral geometry in the solid state. The complex also exists as conformers with trans-octahedral geometry in aqueous media. The active species formed after dissolution in DMSO showed anticancer and antimetastatic behavior in human lung cell line A549 with moderate binding (Kaca. 105 M−1) to bovine serum albumin (BSA). The interaction through hydrogen bonding and van der Waals forces resulted in a spontaneous process. Site marker competitive experiments showed binding sites for chrysin mainly located in site II (subdomain IIIA) and in site I (subdomain IIIA) for the complex. FT-IR spectral measurements showed evidences of the alterations of protein secondary structure in the presence of chrysin and VIVO(chrys)2.

Binding study of florfenicol with DNA by multi-spectroscopy and molecular docking techniques

The investigation on the binding interaction mechanism of florfenicol with DNA in vitro was reported via multi-spectroscopy measurements and molecular docking approach in the present work. The static fluorescence quenching process accompanied with non-radiation energy transfer between florfenicol and DNA was confirmed by Stern-Volmer analysis, lifetime measurement and fluorescence resonance energy transfer study. Florfenicol was the A-T rich minor groove binding agent of DNA. Various experiments including site marker competitive experiments, viscosity measurements, helix melting studies, ionic strength measurements, thermal denaturation experiments, circular dichroism (CD) spectra measurements and UV–vis spectra characteristics were carried out to disclose the interaction mode. In addition, molecular docking stimulated appropriate binding orientation to further prove above experimental results. The binding constants were 6.92 × 103, 6.21 × 103 and 5.61 × 103 M−1 at 291, 301 and 311 K respectively and the binding distance r0 was 5.30 nm. Thermodynamic parameters, such as ΔHӨ =−7.90 kJ mol−1 and ΔSӨ = 46.39 J mol−1 K−1, showed that electrostatic attraction was the key force in the binding.

Exploring the mode of binding between butylated hydroxyanisole with bovine serum albumin: Multispectroscopic and molecular docking study

Considering the harm of BHA on humans, thorough research of the effect of BHA on the structure of serum albumin is necessary. The binding mechanisms of BHA with bovine serum albumin (BSA) and the effects of other three food additives (butylated hydroxytoluene, benzoic acid and citric acid) on BHA-BSA system were researched by multispectroscopy and molecular docking. The fluorescence quenching experiment results showed that the fluorescence quenching mechanism of BSA by BHA was static quenching. The binding constant ((5.70 ± 0.38) × 103 M−1 at 298 K) and thermodynamic parameters (ΔH = 110.8 ± 2.91 kJ·mol−1 and ΔS = 443.3 ± 9.30 J·mol−1·K−1) indicated that BHA and BSA formed a relatively stable complex through hydrophobic interaction. Three-dimensional fluorescence spectra confirmed the conformation changes of BSA due to the binding of BHA. Site marker competitive experiments and molecular docking proved that BHA could bind BSA into site I in subdomain IIA. The results of molecular docking showed that BHA formed hydrophobic interactions with amino acid residues (Ala290, Leu237, Leu259, Ile263 and Ile289). The presence of other food additives weakened the binding of BHA to BSA.

Assessment on the binding characteristics of residual marbofloxacin in animal-derived food to bovine/human serum albumin by spectroscopy and molecular modelling.

To assess the toxicity of residual marbofloxacin from animal-derived food, the interaction characteristics of marbofloxacin to bovine/human serum albumins (BSA/HSA) were explored using spectroscopic methods combined with molecular modelling. According to fluorescence spectra and time-resolved fluorescence spectra measurements, quenching of BSA/HSA fluorescence induced by marbofloxacin was characterized as static quenching. A 1:1 ground-state complex of marbofloxacin to BSA/HSA was formed with binding constant (Ka ) 1.66 × 104 /9.74 × 103 M-1 at 291 K. The location of marbofloxacin binding at site I within BSA/HSA was clarified by site marker competitive experiments. Molecular modelling demonstrated that the binding region for marbofloxacin to BSA and HSA were at site I with the lowest binding free energies of -22.86 and -21.60 kJ mol-1 , respectively. Hydrogen bonds and van der Waals forces were dominantly involved in the spontaneous binding. Nonradiation energy transferred from BSA and HSA to marbofloxacin, due to the close distance (r0 ) between marbofloxacin and Trp residues of BSA (4.28 nm) and HSA (3.34 nm). As explained by circular dichroism (CD) spectra, an increased BSA/HSA α-helix structure was observed after binding to marbofloxacin. Ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectra suggested that conformation of the two proteins was altered by marbofloxacin.

Investigation on the interaction of brazilin with bovine serum albumin using multi-spectroscopic and computational methods: Exploring the binding mechanism and inhibitory effect on amyloid aggregation

Free and active concentration of drug in the blood plays a vital role in the disease treatment. Binding of drug to serum albumin, an important transport protein in plasma, usually improves its aqueous solubility, stability and bioactivity. Brazilin (Bra) is a natural compound extracted from Caesalpinia sappan with numerous pharmacological applications, while the behavior of Bra in circulatory system is still unknown. Therefore, in the present work, we firstly examined the interaction of Bra with bovine serum albumin (BSA) using various spectroscopic and computer simulation techniques. The static quenching mechanism and moderate binding affinity (2.18 × 104 M−1 at 298 K) were obtained through fluorescence spectroscopic measurement. Thermodynamic analysis results indicated that hydrophobic interactions and hydrogen bonds played a major role in the binding process. Site marker competitive experiments and molecular simulation suggested that the probable Bra-binding position was located in subdomain IIA of BSA. UV–vis absorption, circular dichroism (CD) and dynamic light scattering (DLS) experiments suggested the conformational changes of BSA induced by Bra. Additionally, the potential of Bra to inhibit the formation of amyloid fibrils, in vitro, has also been analyzed. Thioflavin T (ThT) binding, 8-anilino-1-naphthalenesulfonic acid (ANS) binding, Congo red binding, DLS, CD, fluorescence microscopic image, atomic force microscopy (AFM), size-exclusion chromatography (SEC) and native gel-electrophoresis methods were used, and all confirmed the anti-amyloidogenic behavior of Bra. In short, our findings have provided some important insights into the binding mechanism of Bra with BSA, and would be helpful in designing suitable agents against amyloid diseases.

Investigation of the binding interactions between 17α-ethinylestradiol with bovine serum albumin by multispectroscopy

To understand the effect of 17α-ethinylestradiol (EE2) on the conformation changes of bovine serum albumin (BSA), the binding mechanisms of EE2 with BSA were investigated by fluorescence spectroscopy, time-resolved fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, UV–visible spectroscopy, circular dichroism (CD) spectroscopy and molecular docking. The quenching constants, binding constants, the number of binding sites, thermodynamic parameters, binding distance and the secondary structure changes of BSA were determined. The results of fluorescence quenching experiment suggested that the fluorescence quenching of BSA by EE was due to the formation of complex through static quenching, which was also confirmed by time-resolved fluorescence measurements. The thermodynamic parameters indicated that the binding of EE2 to BSA was driven mainly by van der Waals forces and hydrogen bonding. The conformation alterations of BSA upon EE2 binding were studied by UV–vis spectroscopy and CD spectroscopy. The results of site marker competitive experiments and molecular docking showed that the binding sites of EE2 were mainly located within site I in the subdomain IIA of BSA.

Synthesis of a novel p-hydroxycinnamic amide and its interaction with bovine serum albumin

A novel p-hydroxycinnamic amides ((E)-4-(3-((4-(N-(5-methoxy-pyrimidin-2-yl) sulfamoyl) phenyl)amino)-3-oxoprop-1-en-1-yl)phenyl acetate (MSAP) was synthesized and confirmed by 1H NMR, 13CNMR ESI-MS, elemental analysis and single-crystal X-ray diffraction analysis. The crystal structure of the complex is monoclinic system and space group is P21/c. The interactions between MSAP and bovine serum albumin (BSA) was investigated with fluorescence and UV–Vis spectral analysis and molecular simulation. According to the fluorescence quenching calculation, the corresponding thermodynamic parameters (ΔH, ΔG and ΔS etc.), bimolecular quenching rate constant (kq), apparent quenching constants (Ksv) and binding sites number (n) were calculated. The distances (r) between BSA and its derivatives were obtained according to Foster’s non-radioactive energy transfer theory. Conformational changes in BSA were observed from synchronous fluorescence spectra. Furthermore, the site marker competitive experiments indicated that the binding of compounds to BSA primarily took place in site I (sub-domain IIA).

Re-assembled oleic acid-protein complexes as nano-vehicles for astaxanthin: Multispectral analysis and molecular docking

In the case of carotenoids, lipid delivery systems are superior in promoting intestinal absorption, while biopolymer-based delivery systems (e.g. protein) have advantages in biocompatibility, biodegradability and controlled release. Therefore, based on the multi-ligand binding properties of protein, we proposed fatty acid-protein complexes as nano-vehicles for encapsulating astaxanthin (ASTA). In this work, oleic acid (OA)–bovine serum albumin (BSA) complexes were fabricated by a pH-driven method. Under the optimized conditions, after OA binding to BSA at a molar ratio of 4:1, slight changes of protein secondary structure appeared and it's surface hydrophobicity increased from 14490 to 25324. When the binding molar ratios of ASTA to BSA ranged from 0.5:1 to 5:1, the ASTA–OA–BSA aqueous dispersions showed better clarity than that of ASTA–BSA. Compared with OA-free BSA, improved encapsulation efficiency and loading capacity were also observed for OA–loading BSA. Based on the fluorescence spectra and Stern–Volmer equation analysis, ASTA binding to the OA-loading BSA displayed a static manner mainly through hydrogen binding and van der Waals interactions. According to the results of site marker competitive experiments and molecular docking, the subdomains IIA and IIIA of BSA should be the acceptor sites of ASTA, and the subdomain IIIA was indicated to be the preferential one. Interestingly, OA as a protein ligand can dramatically enhanced the binding capacity of subdomain IIA to ASTA and make it the preferred acceptor site. In conclusion, OA–BSA nanoparticles as a delivery system should be suitable for fat-soluble nutrients such as carotenoids.

Characterization of binding interaction of triclosan and bovine serum albumin

Triclosan (TCS) is widely used in personal care products and acts as an antibacterial agent. Residues of TCS may have potential effects on the human health. In this article, the interaction between TCS and bovine serum albumin (BSA) has been characterized by using multi-spectroscopic approaches and molecular docking method under physiological conditions. Thermodynamic investigations revealed that TCS spontaneously bound to a binding site of BSA and hydrogen bonds played a dominant role in this process. The site marker competition experiments indicated that TCS bound at site II (subdomain IIIA) of BSA, which was well substantiated by molecular docking. The binding of TCS further led to changes of conformation and microenvironment of BSA. This work explored the interaction of TCS with BSA, which might be beneficial for evaluating the binding mechanism of other environmental pollutant at molecular level.

Evaluation the binding of chelerythrine, a potentially harmful toxin, with bovine serum albumin

Chelerythrine (CHE), a benzophenanthridine alkaloid, is usually used as a nutritional and functional additive in variety of health foods. However, it should be paid enough attention because of its potential toxicity to human health. In this work, the binding mechanism of CHE with bovine serum albumin (BSA) was systematically investigated with spectroscopic approaches. The results showed that the mixture of BSA with CHE could spontaneously cause the formation of BSA-CHE complex through electrostatic interaction under simulative physiological conditions (0.01 mol L−1 Tris-HCl, 0.015 mol L−1 NaCl, pH = 7.4). Site marker competitive displacement experiments exhibited that CHE was primarily bound to the hydrophobic pocket of the site II (subdomain IIIA) of BSA. It has been reported that the binding of small functional molecules to serum albumins remarkably impacts their absorption, distribution, metabolism, conformation, and excretion features. Therefore, this study might be helpful for human to have an in-depth understanding of the biological effect of CHE in vivo and guide human to take it safely and reasonably.

Synthesis, characterization, DNA/BSA interactions and in vitro cytotoxicity study of palladium(II) complexes of hispolon derivatives

Thirteen novel palladium(II) complexes of the general formula [Pd(bipy)(O,O′-dkt)](PF6), (where bipy is 2,2′-bipyridine and O,O′-dkt is β-diketonate ligand hispolon or its derivative) have been prepared through a metal-ligand coordination method that involves spontaneous formation of the corresponding diketonate scaffold. The obtained palladium(II) complexes have been characterized by NMR spectroscopy, ESI-mass spectrometry as well as elemental analysis. The cytotoxicity analysis indicates that most of the obtained palladium(II) complexes show promising growth inhibition in three human cancer cell lines. Flow cytometry analysis shows complex 3e could promote intracellular reactive oxygen species (ROS) accumulation and lead cancer cell death. And the suppression of ROS accumulation and the rescue of cell viability in HeLa cells by N-acetyl-L-cysteine (NAC) suggest the possible link between the increase in ROS generation and cytotoxicity of complex 3e. Flow cytometry analysis also reveal that complex 3e cause cell cycle arrest in the G2/M phase and collapse of the mitochondrial membrane potential, promote the generation of ROS and lead to tumor cell apoptosis. The interactions of complex 3e with calf thymus DNA (CT-DNA) have been evaluated by UV–Vis spectroscopy, fluorescence quenching experiments and viscosity measurements, which reveal that the complex interact with CT-DNA through minor groove binding and/or electrostatic interactions. Further, the results of fluorescence titration and site marker competitive experiment on bovine serum albumin (BSA) suggest that complex 3e can quench the fluorescence of BSA via a static quenching process and bind to BSA in Sudlow's site II.

Binding interaction of sodium benzoate food additive with bovine serum albumin: multi-spectroscopy and molecular docking studies

Sodium benzoate (SB) is widely used as a preservative in food industry, and bovine serum albumin (BSA) is a major carrier protein similar to human serum albumin (HSA), the study of the binding between the two has great significance on human health. In this paper, we systematically investigated the binding of SB and BSA under the simulated physiological conditions combining with various common analytical methods, e.g., fluorescence, UV–vis absorption, synchronous fluorescence and circular dichroism (CD) spectra, as well as molecular docking method. The fluorescence quenching measurements were respectively carried out at 298 K, 303 K and 308 K using the Stern–Volmer method. The results reveal that ground state SB–BSA complex was formed within the binding constants from 2.02 × 104 to 7.9 × 103 M−1. Meanwhile, the negative values of ΔH0 (− 43.92 kJ mol−1) and ΔS0 (− 111.6 J mol−1 K−1) demonstrated that both the hydrogen binding interaction and van der Waals forces contributed to stabilizing the SB–BSA complex. The site marker competitive experiments show that the SB and BSA bound at site I. Furthermore, the experimental results of UV–vis absorption, synchronous fluorescence and CD spectra indicate that the binding of SB and BSA may change the conformation of BSA. In addition, the molecular docking experiment suggests that hydrogen bond was formed in the interaction between SB and BSA.

Multi-spectroscopic measurements, molecular modeling and density functional theory calculations for interactions of 2,7-dibromocarbazole and 3,6-dibromocarbazole with serum albumin

2,7-Dibromocarbazole (2,7-DBCB) and 3,6-dibromocarbazole (3,6-DBCB) are emerging environmental pollutants, being potentially high risks to human health. In this study, interactions of the two compounds with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated by molecular modeling, density functional theory calculations (DFT) and multispectral techniques. The static quenching interaction deduced in the fluorescence quenching experiment is confirmed by the time-resolved analyses. The interactions of the two compounds with HSA/BSA induce molecular microenvironment and conformation changes, as assessed by synchronous and 3D fluorescence spectra, together with a destruction of polypeptide carbonyl hydrogen bond network by circular dichroism and Fourier transform infrared analyses. The thermodynamic analysis indicated that the spontaneous interaction was hydrogen bonding and hydrophobic forces. The binding constant Ka at 298 K was 3.54 × 105 M−1 in HSA-2,7-DBCB, 6.63 × 105 M−1 in HSA-3,6-DBCB, 1.32 × 105 M−1 in BSA-2,7-DBCB and 2.17 × 105 M−1 in BSA-3,6-DBCB. These results indicates that 3,6-DBCB binds HSA/BSA more strongly than 2,7-DBCB, which was estimated with DFT calculations. Site marker competition experiments coupled with molecular modeling studies confirmed that both compounds bind HSA/BSA at site I (subdomain IIA). The results suggest that interactions between 2,7-DBCB and 3,6-DBCB with HSA and BSA may affect the normal physiological activities in human and animals.

Spectroscopic and cytotoxicity studies on the combined interaction of (-)-epigallocatechin-3-gallate and anthracycline drugs with human serum albumin.

The interactions of (-)-epigallocatechin-3-Gallate (EGCG) and anthracycline drugs (doxorubicin, DOX and epirubicin, EPI) alone or in combination with human serum albumin (HSA) under physiological condition were studied by fluorescence spectroscopy, UV-vis absorption spectroscopy, circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). The cytotoxic activity of the single drug, combined drugs, and their complexes with HSA against human cervical cancer HeLa cell line was determined by MTT assay. Fluorescence quenching result and difference spectra of UV absorption revealed the formation of static complex between EGCG, DOX, or EPI and HSA. The binding of EGCG with HSA was driven by both enthalpy and entropy while the binding of DOX or EPI was mainly entropy driven. The nature of binding was expounded based on the effect of sodium chloride, tetrabutylammonium bromide, and sucrose which interfere in electrostatic, hydrophobic, and hydrogen bonding interactions, respectively. Site marker competitive experiments combined with synchronous fluorescence spectra showed that these three ligands mainly bound to subdomain IIA of HSA and were closer to tryptophan residues. In EGCG + DOX/EPI + HSA ternary system, the effect of one drug on the binding ability of another drug was discussed. The influences of the individual and combined binding of EGCG and DOX/EPI on the secondary structure and particle size of HSA were investigated by CD spectroscopy and DLS, respectively. Moreover, the synergistic cytotoxicity of EGCG and DOX/EPI as well as their complexes with HSA were discussed. Obtained results would provide beneficial information on the combination of EGCG and anthracyclines in clinic.